WO2011019399A1 - Preservation of blood vessels using phosphodiesterase inhibitors - Google Patents

Abstract

The disclosure is directed, in part, to the use of phosphodiesterase (PDE) inhibitors to prevent, reduce, or reverse the deleterious effects on blood vessels. The disclosure is also directed to the use of PDE inhibitors to stimulate and enhance the growth of blood vessels and to prevent and/or treat cardiovascular diseases and complications involving the vascular systems.

Description

PRESERVATION OF BLOOD VESSELS USING PHOSPHODIESTERASE

INHIBITORS

RELATED APPLICATIONS

This application claims the benefit under 35 U. S. C. § 1 19(e) of U.S. provisional application serial number 61/233,714, filed August 13, 2009, the disclosure of which is incorporated by reference herein in its entirety.

FEDERALLY SPONSORED RESEARCH

The invention was made with government support under Grant No.

RO1AG02294701 -A1 awarded by the National Heart, Lung, and Blood Institute (NHLBI) of the National Institutes of Health (NIH). The Government has certain rights in the invention.

BACKGROUND OF INVENTION

Deleterious effects on blood vessels, especially collateral blood vessels, caused by various factors, including aging, contribute to dysfunction of vascular reactivity (including collateral dysfunction) thereby increasing cardiovascular diseases and complications. It would be beneficial to have additional novel approaches to protect against and/or to reduce the deleterious effects on blood vessels.

SUMMARY OF INVENTION

Described herein are phosphodiesterase (PDE) inhibitors and their use to protect blood vessels from deleterious effects. In one embodiment, the disclosure provides a method of preventing, reducing or reversing one or more deleterious effects on a blood vessel in a subject (individual), the method comprising administering, to a subject (individual) in need thereof, over an extended period of time, an amount of a phosphodiesterase (PDE) inhibitor sufficient to prevent, reduce or reverse one or more deleterious effects on a blood vessel in the subject (individual).

In one aspect the disclosure provides a method of preventing, reducing or reversing one or more deleterious effects on a blood vessel in a subject, the method comprising administering to a subject in need thereof an effective amount of a phosphodiesterase (PDE) inhibitor over an extended period of time to prevent, reduce or reverse one or more deleterious effects on a blood vessel in the subject. In one aspect the disclosure provides a use of a phosphodiesterase (PDE) inhibitor for preventing, reducing or reversing one or more deleterious effects on a blood vessel in a subject in need thereof in an effective amount for administration over an extended period of time. In one aspect the disclosure provides a use of a phosphodiesterase (PDE) inhibitor in the preparation of a medicament for preventing, reducing or reversing one or more deleterious effects on a blood vessel in a subject in need thereof, wherein the medicament is for administration of the phosphodiesterase (PDE) inhibitor over an extended period of time.

In some embodiments of the methods and the uses presented herein, the method and uses further comprise increasing nitric oxide levels in the subject, for instance by increasing the activity of down-stream products of eNOS (endothelial Nitric Oxide Synthetase). In some embodiments, the nitric oxide levels in the subject are increased through aerobic exercise. In some embodiments, the nitric oxide levels in the subject are increased through the administration of a nitric oxide inducer. In some embodiments of the methods and the uses presented herein, protective effects of eNOS in the subject are increased through the administration of an agent that decreases the degradation of products of the eNOS system that are vascular protective. In some embodiments, the preventing, reducing or reversing one or more deleterious effects on a blood vessel comprises suppressing a loss of arterioles or collaterals, suppressing a loss in collateral dysfunction or suppressing a decrease in collateral flow recovery. In some embodiments, the subject is otherwise free of indications for treatment with the phosphodiesterase (PDE) inhibitor. In some embodiments, the deleterious effect is caused by aging, hypercholesterolemia, hypertension, hyperlipidemia, obesity, diabetes mellitus, smoking, genetic predisposition or lifestyle. In some embodiments, the blood vessel is present in at least one of the following: heart, brain, extremity, or kidney. In some embodiments, the subject is at risk of a cardiovascular condition. In some embodiments, the cardiovascular condition is heart attack, stroke, renal failure, claudication, loss of leg, hypercholesterolemia, hypertension, hyperlipidemia, obesity, diabetes mellitus, smoking, or genetic predisposition. In some embodiments, the cardiovascular conditions are conditions that can be exacerbated or caused by aging. In some embodiments, the cardiovascular condition is caused by atherosclerosis. In some embodiments, the phosphodiesterase (PDE) inhibitor is selected from the group consisting of Vinpocetine, EHNA (erythro-9-(2-hydroxy-3-nonyl)adenine), Cilomilast, Etazolate, Glaucine, Ibudilast, Mesembrine, Rolipram, Acetildenafil, Avanafil, Sildenafil, Tadalafil, Udenafil, Vardenafil, Milrinone and Amrinone.

In one aspect the disclosure provides a method for preserving or increasing the functional capacity of a blood vessel in a subject, the method comprising administering to a subject in need thereof an effective amount of a phosphodiesterase (PDE) inhibitor over an extended period of time to preserve or increase the functional capacity of the blood vessel in the subject. In one aspect the disclosure provides a use of a

phosphodiesterase (PDE) inhibitor for preserving or increasing the functional capacity of a blood vessel in a subject in need thereof in an effective amount for administration over an extended period of time. In one aspect the disclosure provides a use of a

phosphodiesterase (PDE) inhibitor in the preparation of a medicament for preserving or increasing the functional capacity of a blood vessel in a subject in need thereof wherein the medicament is for administration over an extended period of time. In some embodiments, of the methods and uses presented herein the method and use further comprise increasing nitric oxide levels in the subject, such as by increasing the activity of key down-stream products of eNOS that are vascular-protective. In some

embodiments, the nitric oxide levels in the subject are increased through aerobic exercise. In some embodiments, the subject is either otherwise free of indications for treatment with the phosphodiesterase (PDE) inhibitor, or uses PDE inhibitors on a less than regular basis for such a condition as erectile dysfunction. In some embodiments, the blood vessel is present in at least one of the following: heart, brain, extremity or kidney. In some embodiments, the phosphodiesterase (PDE) inhibitor is selected from the group consisting of Vinpocetine, EHNA (erythro-9-(2-hydroxy-3-nonyl)adenine), Cilomilast, Etazolate, Glaucine, Ibudilast, Mesembrine, Rolipram, Acetildenafil, Avanafil,

Sildenafil, Tadalafil, Udenafil, Vardenafil, Milrinone and Amrinone.

In one aspect the disclosure provides a method for reducing the incidence and/or severity of at least one of the following conditions: heart attack, stroke, renal failure, claudication, loss of limb or vascular occlusions, the method comprising administering to a subject in need thereof an effective amount of a phosphodiesterase (PDE) inhibitor over an extended period of time to reduce the incidence and/or severity of at least one of the conditions selected from the group consisting of heart attack, stroke, renal failure, claudication, loss of limb and vascular occlusions. In one aspect the disclosure provides a use of a phosphodiesterase (PDE) inhibitor for reducing the incidence and/or severity of at least one of the following conditions: heart attack, stroke, renal failure, claudication, loss of limb or vascular occlusions, in a subject in need thereof in an effective amount for administration over an extended period of time. In one aspect the disclosure provides a use of a phosphodiesterase (PDE) inhibitor in the preparation of a medicament for reducing the incidence and/or severity of at least one of the following conditions: heart attack, stroke, renal failure, claudication, loss of limb or vascular occlusions, in a subject in need thereof wherein the medicament is for administration over an extended period of time. In some embodiments of the methods and use presented herein the method and use further comprise increasing the levels of NO in the subject, such as by increasing in the subject the activity of key down-stream products of eNOS that are vascular-protective. In some embodiments, the nitric oxide levels in the subject are increased through aerobic exercise. In some embodiments, the blood vessel is present in at least one of the following: heart, brain, extremity or kidney. In some embodiments, the

phosphodiesterase (PDE) inhibitor is selected from the group consisting of Vinpocetine, EHNA (erythro-9-(2-hydroxy-3-nonyl)adenine), Cilomilast, Etazolate, Glaucine, Ibudilast, Mesembrine, Rolipram, Acetildenafil, Avanafil, Sildenafil, Tadalafil, Udenafil, Vardenafil, Milrinone and Amrinone.

This disclosure is related, in one embodiment, to the use of phosphodiesterase (PDE) inhibitors to protect blood vessels from deleterious effects. In a particular embodiment, a phosphodiesterase (PDE) inhibitor is administered in a method of preventing, reducing, or reversing one or more deleterious effects on a blood vessel. The method comprises administering, to a subject in need thereof, an effective amount of a PDE inhibitors, over an extended period of time, to prevent, reduce, or reverse the deleterious effects on the blood vessel.

The deleterious effect(s) may be caused by aging, hypercholesterolemia, hypertension, hyperlipidemia, obesity, diabetes mellitus, smoking, genetic predisposition or lifestyle. In some embodiments, the deleterious effect is in a blood vessel present in at least one of the following: the heart, brain, extremity, or kidney. In some embodiments, the subject is at risk of developing a cardiovascular condition, such as heart attack, stroke, renal failure, claudication or loss of a leg. The cardiovascular condition may be heart attack, stroke, renal failure, claudication, or loss of a leg. These conditions can be exacerbated or caused by aging, hypercholesterolemia, hypertension, hyperlipidemia, obesity, diabetes mellitus, smoking, or genetic

predisposition.

Also described herein is a method of preserving or increasing the functional capacity of a blood vessel in a subject. The method comprises administering, to a subject in need thereof, an effective amount of a PDE inhibitor, over an extended period of time, to enhance the survival or to increase the functional capacity of the blood vessel. In some embodiments, the blood vessel is present in at least one of the following: the heart, brain, extremity or kidney.

A further embodiment is a method of reducing the incidence and/or severity of at least one of the following conditions: heart attack, stroke, renal failure, claudication, loss of limb or vascular occlusion is provided. The method comprises administering an effective amount of a PDE inhibitor, to a subject in need thereof, over an extended period of time, to reduce the incidence and/or severity of the condition.

In some embodiments, the subject does not present with symptoms or an indication that he or she is in need of treatment with a PDE inhibitor. In some embodiments, in addition to being treated with a PDE inhibitor, the subject undertakes sufficient aerobic exercise, typically on a regular basis, to increase his/her heart rate and blood flow, for example.

PDE inhibitors that can be used in the methods and compositions described herein include, but are not limited to, Vinpocetine, EHNA (erythro-9-(2-hydroxy-3- nonyl)adenine), Cilomilast, Etazolate, Glaucine, Ibudilast, Mesembrine, Rolipram, Acetildenafil, Avanafil, Sildenafil, Tadalafil, Udenafil, Vardenafil, Milrinone and Amrinone. In some preferred embodiments, the PDE inhibitor is Sildenafil.

As used herein, "a" phosphodiesterase (PDE) inhibitor refers to "at least one" or "one or more" phosphodiesterase (PDE) inhibitors.

These and other aspects of the disclosure, as well as various advantages and utilities will be apparent with reference to the Detailed Description. Each aspect of the disclosure can encompass various embodiments, as will be understood. All documents identified in this application are incorporated in their entirety herein by reference.

BRIEF DESCRIPTION OF DRAWINGS

The figures are illustrative only and are not required for enablement of the invention disclosed herein.

Figures IA- I B are graphs showing, respectively, the blood flow recovery in C57bl/6 female mice at different ages subjected to femoral artery ligation and extirpation and necrosis. A) Blood flow recovery for 4, 10 and 14 month-old female C57BL/6J mice, as measured using laser Doppler flow imaging. Flow recovery is impaired in 14 vs. 4 month-old mice (*p<0.001). 10-month old mice have intermediate values. B) Necrosis score. Necrotic score is greater in the ischemic calf in 14 vs. 4 month-old mice (*p< 0.001).

Figures 2A-2D indicate the fold change in expression of MMP9 (mRNA and protein), HIF-lalpha (mRNA) and SDF-I (mRNA) up to 7 days post-ischemia induction.

Figures 3 A - 3C indicate changes in VEGF mRNA and protein and eNOS protein levels following femoral artery ligation and extirpation. Western blot demonstrates that VEGF protein is elevated in young mice at both days 3 and day 7 post surgery.

Figures 4A - 4B are graphs that show changes in CD26 mRNA and percentage of BM cells staining positive for CD26 in young vs. old mice before and after femoral artery ligation and extirpation.

Figure 5 is a graph that shows the effects of age of bone marrow (BM) transplantation recipient and donor on collateral flow.

Figures 6A - 6B show the effects of aging on angiogenesis using a Matrigel plug angiogenesis assay in young and old male C57B1/6 mice.

Figures 7A - 7B depict brain collaterals in young and old mice. Figures 7C - 7E are graphs indicating the tortuosity index, collateral diameter and collateral resistance of six randomly chosen collaterals in young and old mice. Data indicate that aging- impaired in collaterogenesis. Brain collaterals (pial circulation) in young (3 month old) vs. aging (16 month old) mice.

Figure 8 is a graph showing the blood flow recovery in young and old C57bl/6 male mice subjected to femoral artery ligation. Figures 9A- 9E depict hindlimb collaterals in 9A) young mouse thigh, 9B) older mouse thigh, 9C) eNOS KO mouse thigh. Analysis was then carried out by batching the groups into vessels <24 μm (Fig 9D) and >24 μm (Fig 9E).

Figures 1OA - 1OB are graphs showing the number of small arteries (collaterals) in young and old mice in the thigh and calf.

Figures 1 IA - 1 IB are graphs showing the number of small arteries (collaterals) in young, old and eNOS knock out mice in the thigh and calf.

Figures 12A - 12B show phosphorylated and total eNOS protein as determined by Western blotting (A). The ratio of phosphorylated/total eNOS protein levels was significantly (*p=0.015) lower in old vs. young mice (B).

Figure 13A shows microvascular dropout in mouse hindlimb. As mice age there is a dropout of small arteries. Left two graphs: micro-CT. Third graph: histologically determined α-SMC-actin-positive vessel density in collateral mid-zone of adductor thigh region.

Figure 13B shows perfusion immediately after femoral artery ligation. At this time, prior to any collateral growth or remodeling, the flow is carried by native preexisting collaterals, and is therefore an index of native collateral conductance, which is determined mainly by collateral number, diameter, and length. By as early as 16 months of age, collateral flow immediately post-occlusion is reduced. This could be caused by either: a) failure of shear-stress signaling pathways to inhibit collateral smooth muscle tone after ligation of the major conduit vessel they parallel, b) aging-induced rarefaction, c) narrowing of native collateral diameter, or d) lengthening of the native collaterals. Our data show that decreases in collateral number and diameter contribute to the decreased flow.

Figure 13C shows that aging tends to decrease native hindlimb collateral diameter and markedly impairs positive remodeling in hindlimb following femoral artery ligation. Although the decrease in hindlimb collateral diameter is not significant, it follows the significant aging-induced decrease in collateral diameter present in the pial circulation.

Figure 14A shows that age-related changes in collateral diameter, number, length, and calculated relative resistance in the mouse brain. Aging led to significant deleterious changes in collateral diameter, number, and length, which resulted in marked increases in resistance.

Figure 14B shows the effects of MCA occlusion on collateral remodeling. Left panel shows pial collateral (arrowhead) on the non-ligated side in a young mouse.

Middle panel shows comparable collaterals on the ligated side, 3 days after MCA ligation. Right panel shows mean increase in collateral diameter following MCAO in young vs. old mice. Aging inhibits positive remodeling of brain collaterals following conduit artery occlusion.

Figure 14C shows the effects of aging on volume of cerebral infarction following MCAO. The aging-induced abnormalities leading to increased collateral resistance (See Fig 14A) were associated with a 2.8-fold increase in the volume of infracted brain tissue.

Figure 15 shows the effects of aging on eNOS protein levels and eNOS activation, as determined by p-eNOS (ser-1 177) levels in the non-ischemic calf of young-3 months (Y) and old 24 months (O) mice.

Figure 16 shows that the ECs (passage 5) derived from old (24 months-old) vs. young (3 months-old) mice are more sensitive to an apoptotic stimulus— TNFα (left panel). Moreover, exposure of the cells to sodium nitroprusside (SNP) prior to addition of TNFα rescues the cells such that the magnitude of apoptosis in old vs. young cells is similar.

Figure 17 shows the increased sensitivity of "old" SMCs to undergo H₂O₂- mediated apoptosis (top panel), and its attenuation by the PDE5-I inhibitor tadalafil (bottom panel).

Figure 18 shows VASP-levels. Young mice were exercised 5d/wk, 50 min per session for 3 months. They were sacrificed 24 hours after their last bout of exercise and P-VASP levels were determined by western blot.

Figure 19 shows that collateral flow in 22 months old mice, exercising daily for 15 days (-50 min each session), is augmented by tadalafil. Mice received oral tadalafil 3 days after start of exercise daily for 12 days (80μg/day). On day 15 the distal femoral artery was occluded and tadalafil continued. 24 hrs after surgery mice received tadalafil and swam for the last time; several hours later (28 - 30 hrs after femoral artery occlusion), collateral flow was measured. Figure 20 shows time courses showing changes in expression of P-VASP protein in young and old mice following acute occlusion of the femoral artery (FA). Within 24 hours following FA occlusion, the presumed increased flow and sheer stress occurring in the collaterals leads, in young mice (panel A), to increased eNOS signaling, evidenced by an increase in P-VASP in the collateral wall on day 1 post occlusion. P-VASP levels diminish to control (collaterals in the contralateral non-ligated hindlimb) by day 7, a change probably explained by the robust positive collateral remodeling that occurs in the young mice (panel D), which would decrease shear forces. This sequence of beneficial eNOS signaling is markedly deranged in old mice. There is no increase in P-VASP levels in the collaterals of old mice 24 hours after femoral artery ligation (panel B). By day 7 there is actually a trend towards a decrease in P-VASP. These changes are most evident with the data displayed as changes from control (panel C). As expected, the aging-induced dysfunction of eNOS/NO signaling is associated in the old mice with markedly impaired collateral positive remodeling (panel D)— the actual decrease_seen in this group of mice studied (i.e., negative remodeling), was not seen in our studies investigating the cerebral collateral system or the hindlimb of other groups of mice which demonstrated aging-related reduced positive remodeling.

Figure 21 shows that aging is associated with increased oxidative stress.

Nitrotyrosine protein levels were assayed by Western blot of mesenteric arteries obtained from young (3 months) and old (24 months) mice. Nitrotyrosine is formed by nitration of protein tyrosine residues by peroxynitrite, and therefore is a marker for peroxynitrite formation in vivo. These data demonstrate that nitrotyrosine levels are increased in the mesenteric arteries of old mice, indicating increased vascular oxidative stress exists in our model, and implies that eNOS is uncoupled with signaling proceeding preferentially down the vascular deleterious-eNOS.

Figure 22 shows the effects of aging on PDE5 mRNA levels. SMCs harvested from the aortas of young vs. old mice were assayed for PDE5 mRNA. PDE5 expression was markedly increased in SMCs derived from the old mice. DETAILED DESCRIPTION OF THE INVENTION

Described herein are methods for preventing, reducing or reversing the deleterious effects that occur in a subject's blood vessels. These deleterious effects may be the result of aging or of other factors, such as, hypercholesterolemia, hypertension, hyperlipidemia, obesity, diabetes mellitus, smoking, genetic predisposition and/or lifestyle (e.g., level of exercise, diet, alcohol consumption, stress). In some

embodiments, the deleterious effects may occur in one or more blood vessels present in at least one of the following: the heart, brain, extremity and kidneys. The methods described herein may also be used to enhance the survival and stimulate the growth of blood vessels and to improve their function.

One embodiment relates to methods for preventing, reducing, or reversing the deleterious effects on a blood vessel in a subject. In some embodiments, the methods comprise increasing the vascular-beneficial activity of eNOS (endothelial Nitric Oxide Synthetase) expression. In some embodiments, the methods comprise increasing the levels of eNOS signaling. In some embodiments, the methods comprise administering an effective amount of a phosphodiesterase (PDE) inhibitor. In some embodiments, the PDE inhibitor is administered for an extended period of time. In some embodiments, the methods comprise increasing nitric oxide (NO) levels in a subject. In some

embodiments, NO levels are increased through the administration of a nitric oxide inducer. In some embodiments, NO levels are increased through aerobic exercise, which can result in the increase in the subject's heart rate and blood flow. In some

embodiments, the methods comprise increasing the levels of eNOS, increasing eNOS signaling or both (increasing eNOS signaling in combination with increasing the levels of NO in a subject). In some embodiments, the methods comprise administering an effective amount of a PDE inhibitor to a subject who undertakes aerobic exercise, typically on a regular basis.

It is shown herein that aging decreases collateral flow recovery. In addition, it is shown herein that the protective effects of eNOS in the subject are decreased with aging. In addition to the aging-induced impaired activity of key down-stream products of eNOS that are vascular-protective and dysfunctional effects of an aging-induced eNOS uncoupling (increased nitrosylation of proteins), aging also impairs eNOS signaling. Phosphorylation of eNOS at serine 1 177 (Ser-1 177) is critical for eNOS signaling and for NO generation, and it is shown herein that phosphorylation at this site is reduced by aging. Importantly, the ratio of phosphorylated eNOS to total eNOS was also significantly lowered upon aging, indicating that decreased eNOS signaling was due not only to a decrease in the level of eNOS protein but also to an impaired mechanism involved in SeM 177 phosphorylation. eNOS/NO signaling plays an important role in vascular function, including preventing aging-related endothelial cell apoptosis. It is shown herein that the detrimental effects aging has on the levels of eNOS and eNOS signaling compromises the integrity of the vascular system. Aging is associated with a loss of arterioles as aging mice have fewer arterioles and pre-existing collaterals in their hindlimb when compared to young wild type mice. The causal role of eNOS in the aging-related dropout of microvessels was confirmed by demonstrating that young eNOS KO (knock-out) mice have fewer pre-existing collaterals in their hindlimb when compared to young wild type mice. Furthermore, the same pattern of aging-related collateral dropout was found in the brain. Young eNOS KO mice in fact show the same pattern as aging wild type mice. Thus, the impairment of the NO pathway contributes to aging-induced loss of collaterals.

Described herein are methods for preventing, reducing, or reversing the deleterious effects on a blood vessel in a subject, comprising increasing the levels of eNOS (endothelial Nitric Oxide Synthetase) expression and/or decreasing degradation of key down-stream products of eNOS that are vascular-protective.

The methods presented herein provide a new therapeutic strategy that attenuates the impairment of the eNOS/NO system that causes aging-induced deleterious effects on collateral phenotype and function. By administering agents that prolong and enhance the vascular activity of NO and/or its downstream products, the deleterious effects that contribute to aging-related decreased activity of the eNOS/NO system are suppressed, reduced or reversed. The vascular activity of the eNOS/NO system is initiated through the activation of guanylate cyclase, which results in the increase of cGMP levels, causing smooth muscle cell relaxation and thereby vasodilation. Levels of cGMP are deceased through the action of phosphodiesterases (PDEs) which degrade cGMP. Thus, suppressing the activity of PDE results in an increase in the level of cGMP and thus in increased NO signaling. Chronic administration of a PDE inhibitor can therefore overcome the eNOS/NO mediated aging-related deterioration in collateral function. In some embodiments, the administration of a PDE inhibitor suppresses the effects of an increased oxidative state in a blood vessel, such as that induced by aging, thereby providing an additional protective role against deleterious effects on a blood vessel.

In some embodiments, the methods comprise combination approaches, such as administering PDE inhibitors and increasing NO levels. PDE inhibitors act by decreasing the degradation of NO-mediated increased cGMP. However, unless sufficient NO levels are present to increase cGMP, there is not enough cGMP available for an agent that acts by decreasing cGMP degradation to manifest biologically relevant activity. NO levels can be increased be a variety of methods. In some embodiments, NO levels are increased through the administration of an NO inducer. In some embodiments, NO levels are induced through aerobic exercise. Aerobic exercise has been shown to augment the endothelial eNOS/NO system (Hambrecht et al., Circulation 2003

107:3152-3158). In addition, exercise has been shown to increase eNOS protein expression (Iemitsu et al., Am J Physiol Hear Circ Physiol 2006, 291 : H 1290- 1298).

In one embodiment a preventive method is provided to suppress the negative vascular effects associated with aging by combining pharmacologic enhancement of NO (through the administration of PDE inhibitors) with an appropriate stimulus to the endothelium lining of the collaterals (through an increase ion NO levels, for instance by aerobic activity). Thus, the combined administration of a PDE- inhibitor and regular exercise, initiated early during the course of the "disease" (aging), will prevent or retard the deleterious effects of aging on collateral function.

In one aspect, the disclosure provides methods for preventing, reducing or reversing one or more deleterious effects on a blood vessel in a subject. As used herein, a "deleterious effects on a blood vessel" is any effect that interferes with the optimal functioning of a blood vessel. Deleterious effects on a blood vessel include, but are not limited to, loss of arterioles, the reduction in pre-existing collaterals, the dropout of microvessels, deterioration of collateral function, collateral dysfunction, impaired collaterogenesis, dropout of pre-existing collateral vessels and/or vasculature, increased tortuosity, decreased diameter of collateral vessels, impaired capacity to dilate, impaired VEGF expression, impaired response to ischemia, decreased vasolidation, decreased collaterogenesis, and a reduction in collateral blood flow recovery in response to arterial occlusions.

"Deleterious effects on blood vessels" are manifested, for example, by a reduction in collateral blood flow recovery in response to arterial occlusions, impaired collaterogenesis, drop-out of pre-existing collateral vessels and/or vasculature, increased tortuosity and decreased diameter of such vessels, impaired capacity to dilate, impaired VEGF expression and/or reduced eNOS levels or vascular protective eNOS activity.

As used herein, the term blood vessel means any channel in which blood circulates or flows in a subject. Blood vessels includes but are not limited to, arteries, arterioles, capillaries, veins, venules and collaterals thereof. In some embodiments, the blood vessel has a diameter of less than about 5 mm, 2 mm, 1 mm, 500 μm, 100 μm, 50 μm, 25 μm, 15 μm, 10 μm, 5 μm, 1 μm, or less. The blood vessel can be located in any part of the subject. In some embodiments, the blood vessel is in the heart, brain, extremity or kidney.

In one aspect, the disclosure provides methods for preserving or increasing the functional capacity of a blood vessel in a subject. The functional capacity of a blood vessel, as used herein, is any functionality that can be ascribed to a blood vessel.

Functional capacities include but are not limited to expression of specific proteins, ability to reaction to injury, vasodilation, collaterogenesis, capacity to dilate and collateral flow recovery.

As used herein, the methods for preventing are prophylactic. As used herein, the methods for reducing, preserving, increasing may be prophylactic and/or therapeutic. It should be appreciated that prophylactic treatment may result in a partial prevention, e.g., a percentage reduction, for example about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or higher or lower or intermediate percentages of reduction of the deleterious effect on a blood vessel compared to not undergoing the methods of the disclosure. However, in some embodiments, a prevention may be complete (e.g., a 100% reduction or about a 100% reduction based on an assay).

As used herein, the methods for reducing, preserving, increasing may be prophylactic and/or therapeutic. As used herein, the methods for reversing are therapeutic. The therapeutic methods can result in a partial or complete treatment. Thus, the therapeutic methods disclosed herein may result in a complete reversal of the deleterious effect on the blood vessel. In some embodiments, the therapeutic methods may result in a partial reversal of the deleterious effect, e.g., a percentage change, for example about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or higher or lower or intermediate percentages of reduction of the deleterious effect on a blood vessel compared to not undergoing the therapeutic methods of the disclosure.

The methods of preventing, reducing, preserving, increasing and reversing presented herein may effect one or more of the deleterious effects, e.g., a method of reducing may result in a 10% suppression of loss of arterioles and a 50% suppression of collateral loss. It should be appreciated that the methods presented herein may also result in a desired response for one deleterious effect, while having an undesired response for another deleterious effect (e.g., resulting in the suppression of the loss of arterioles while resulting in an increase in tortuosity).

In one aspect the disclosure provides methods for reducing the incidence and/or severity of at least one of the following conditions: heart attack, stroke, renal failure, claudication, loss of limb or vascular occlusions. Reducing the incidence, as used herein, means a decrease in the number of occurrence of an event or reducing the chance that an incident may occur. It should be appreciated that reducing the incidence means both reducing the risk (as can be expressed in percentages) and reducing the number of occurrences. For instance, the methods provided herein may result in a decrease in the chance of getting a stroke (e.g., a reduction from 80 % to 50%) or may result in a decrease in the number of occurrences of an event (e.g., a vascular occlusion once every 10 years in stead of a vascular occlusion once every 2 years). The chance of having the condition can be determined by a physician, for instance by performing diagnostic assays, which could include, for example, exercise testing, angiographic procedures, performing CT scanning of the vasculature, etc. Reducing the severity as used herein means reducing the deleterious effects of an event. Reducing the severity as used herein includes both reducing the severity of the condition when it occurs (e.g., reducing the severity of the stroke or the heart attack) and reducing the severity of the consequences of the condition (e.g., reducing the severity of the consequences of the stroke or heart attack). In one aspect the disclosure provides methods for preventing, reducing or reversing one or more deleterious effects on a blood vessel in a subject, wherein the deleterious effect is caused by aging, hypercholesterolemia, hypertension,

hyperlipidemia, obesity, diabetes mellitus, smoking, genetic predisposition or lifestyle. The relationship between aging, hypercholesterolemia, hypertension, hyperlipidemia, obesity, diabetes mellitus, smoking, genetic predisposition or lifestyle and their deleterious effects on blood vessels is well known. For instance, a subject who has hypertension generally will have a decreased diameter of blood vessels (a deleterious effect) when compared to a normotensive subject. Similarly, as shown herein, aging result in a number of deleterious effect on blood vessels, including the loss of arterioles. A health care professional can determine if a person has hypercholesterolemia, hypertension, hyperlipidemia, obesity, diabetes mellitus or may have a genetic predisposition {e.g., mutations in a gene encoding a protein involved in vascular homeostasis), while the age and smoking habits of a subject will be known or can readily be determined. The relationship between lifestyle and deleterious effects on blood vessels has also been well established. For instance, subjects with a sedentary lifestyle and/or poor diet are more likely to suffer from deleterious effects on blood vessels than subjects with an active lifestyle and healthy diet.

In some aspects, the methods of the disclosure may help prevent, decrease or reverse deleterious effects on blood vessels of subjects that are at risk of a cardiovascular condition or event. Cardiovascular conditions or events include, for example, acute coronary syndrome, myocardial infarction, myocardial ischemia, chronic stable angina pectoris, unstable angina pectoris, stroke, renal failure, transient ischemia attack, claudication, loss of a leg or vascular occlusion(s). These conditions or events may be caused by aging, or by other risk factors including, but not limited to,

hypercholeterolemia, hypertension, hyperlipidemia, obesity, diabetes mellitus, smoking, genetic predisposition or lifestyle.

A subject who has had a primary cardiovascular event is at an elevated risk of a secondary (second) cardiovascular event. In some embodiments, the subject has not had a primary cardiovascular event, but is at an elevated risk of having a cardiovascular event because the individual has one or more risk factors to have a cardiovascular event.

Examples of risk factors for a primary cardiovascular event include: hyperlipidemia, obesity, diabetes mellitus, hypertension, pre-hypertension, elevated level(s) of a marker(s) of systemic inflammation, age, a family history of cardiovascular events, and cigarette smoking. The degree of risk of a cardiovascular event depends on the multitude and the severity or the magnitude of the risk factors that the subject has. Risk charts and prediction algorithms are available for assessing the risk of cardiovascular events in a human subject based on the presence and severity of risk factors. One such example is the Framingham Heart Study risk prediction score. A subject is at an elevated risk of having a cardiovascular event if the subject's 10-year calculated Framingham Heart Study risk score is greater than 5%, 6%, 7%, 8%, 9%, 10%, 1 1%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% or more.

In one aspect the disclosure provides methods for preventing, reducing or reversing one or more deleterious effects on a blood vessel in a subject, methods for preserving or increasing the functional capacity of a blood vessel in a subject and methods for reducing the incidence and/or severity of heart attack, stroke, renal failure, claudication, loss of limb or vascular occlusion in the subject, wherein the methods comprise increasing nitric oxide (NO) levels in a subject, such as by increasing the down-stream products of eNOS that are vascular-protective. In some embodiment the methods comprise increasing nitric oxide levels in the blood vessels, such as by increasing in the subject the activity of key down-stream products of eNOS that are vascular-protective. In some embodiments, the methods are combined with the administration of a PDE inhibitor.

Nitric oxide (NO) is a biological messenger, playing a role in a variety of biological processes. Nitric oxide is biosynthesised endogenously from arginine and oxygen by nitric oxide synthase (NOS) enzymes, including endothelial NOS (eNOS), and by reduction of inorganic nitrate. Platelet derived factors, shear stress, acetylcholine, and cytokines stimulate the production of NO by endothelial nitric oxide synthase (eNOS). eNOS synthesizes NO from the terminal guanidine-nitrogen of L-arginine and oxygen and yields citrulline as a byproduct. NO production by eNOS is also dependent on calcium-calmodulin and other cofactors. Nitric oxide is highly reactive (having a lifetime of a few seconds), yet diffuses freely across membranes. The endothelium of blood vessels use nitric oxide to signal the surrounding smooth muscle to relax, thus resulting in vasodilation and increasing blood flow. Nitric oxide (NO) contributes to vessel homeostasis by inhibiting vascular smooth muscle contraction and growth, platelet aggregation, and leukocyte adhesion to the endothelium.

The level of nitric oxide (NO) in a subject can be determined using a variety of assays. A sample (e.g., blood, plasma or cells) can be taken from a subject and the concentration of nitric oxide determined. One method of assaying involves

electroanalysis, where NO reacts with an electrode to induce a current or voltage change. Another assay is a spin trapping assay in which nitric oxide complexes with iron- dithiocarbamate which allows for subsequent detection of the mono-nitrosyl-iron complex with Electron Paramagnetic Resonance (EPR). Yet another method is provided through the use of fluorescent dye indicators (e.g., 4,5-diaminofluorescein (DAF-2)) which can be used for intracellular measurements.

Methods for increasing nitric oxide levels in subject include both pharmalogical methods (i.e., the administration of one or more NO inducing compounds to a subject) and non-pharmalogical methods (e.g., exercise), and combinations thereof. NO inducing compounds include any compound that, upon administration, indirectly or directly increases the nitric oxide level in a subject. Nitric oxide inducers include compounds that stimulate endogenous NO, elevate levels of endogenous NO in vivo, or are substrates for nitric oxide synthase. Such compounds include, for example, L-arginine, L- homoarginine, and N-hydroxy-L-arginine, including their nitrosated and nitrosylated analogs (e.g., nitrosated L-arginine, nitrosylated L-arginine, nitrosated N-hydroxy-L- arginine, nitrosylated N-hydroxy-L-arginine, nitrosated L-homoarginine and nitrosylated L-homoarginine), precursors of L-arginine and/or physiologically acceptable salts thereof, including, for example, citrulline, ornithine, glutamine, lysine, polypeptides comprising at least one of these amino acids, inhibitors of the enzyme arginase (e.g., N- hydroxy-L-arginine and 2(S)-amino-6-boronohexanoic acid) and the substrates for nitric oxide synthase, cytokines, adenosine, bradykinin, calreticulin, bisacodyl, and

phenolphthalein. Additional nitric oxide inducing compounds include nitroglycerin and amyl nitrates including isobutyl nitrate.

In one aspect the disclosure provides methods for increasing the levels of nitric oxide in a subject through aerobic exercise. The disclosure embraces any form of aerobic exercise including running, walking, swimming, biking, etc. A variety of exercise regimens are contemplated. The exercise regime can include a variety of frequencies including exercising daily, twice daily, every other day, three times a week, two times a week, weekly, biweekly, monthly or any frequency in between. The length of the aerobic exercise may vary and can range from a couple of minutes to multiple hours. Combinations of different lengths and frequencies are contemplated as well (e.g., three exercises a week, one of 30 minutes and two of at least one hour). Exercise regimes can be optimized by determining the amount of nitric oxide produced. A clinician, physician or physiologist can determine the level of nitric oxide in a subject and change the regimen to result in the optimal level of nitric oxide in the subject.

Exercise regimes, as contemplated herein, may be adhered to for a number of days, a number of months, a number of years up to the total lifespan of the subject.

In one aspect, the disclosure provides methods comprising the administration of PDE inhibitors to a subject. A "subject", as used herein, is a human or other vertebrate mammal including, but not limited to, mouse, rat, dog, cat, horse, cow, pig, sheep, goat, or non-human primate. In some embodiments, the subject is male. In some

embodiments, the subject is female. A "subject" is also referred to herein as an

"individual" and the two can be used interchangeably.

A "subject in need thereof (e.g., in need of a method for preventing, reducing or reversing one or more deleterious effects on a blood vessel, a method for preserving or increasing the functional capacity of a blood vessel, or a method for reducing the incidence and/or severity of heart attack, stroke, renal failure, claudication, loss of limb or vascular occulsion), as used herein, means a subject who is identified as being in need of the methods provided herein. For instance, a subject in need of any of these methods is a subject identified as benefiting from any of the methods of the disclosure. A "subject in need thereof is, for example, a human who has had or is susceptible to developing a condition in which his/her blood vessel(s) have been adversely affected or is at risk for such a condition. "Subjects in need thereof include subjects that have hypercholesterolemia, hypertension, hyperlipidemia, obesity, diabetes mellitus, hypertension, pre-hypertension, systemic inflammation, are of advanced age, have a family history of cardiovascular events, are smokers, have a genetic predisposition and/or specific lifestyle (e.g., level of exercise, diet, alcohol consumption, stress).

In some embodiments, the subject is otherwise free of indications calling for treatment with a PDE inhibitor. A subject free of indications calling for treatment with a PDE inhibitor is a subject who has no signs or symptoms calling for treatment with a PDE inhibitor. Indications calling for treatment with a PDE inhibitor are known to those of ordinary skill in the art. Examples of such indications include erectile dysfunction, pulmonary hypertension, high altitude pulmonary edema, asthma, stroke, cerebrovascular disorder (e.g., stroke), and congestive heart failure.

As used herein, a "PDE inhibitor" is a molecule that can inhibit one (or more) isoforms of the enzyme phosphodiesterase, thereby preventing the degradation of intracellular second messengers, such as cAMP and cGMP. The potential use of selective PDE inhibitors as therapeutic agents was predicted as early as 1977 by Weiss and Hait. (Weiss, B. and Hait, W.N. (1977) Ann. Rev. Pharmacol. Toxicol. 17:441-477, 1977). PDE inhibitors as used herein include both non-specific and specific PDE inhibitors. Non-specific PDE inhibitors include caffeine, theophylline and (3-isobutyl- 1-methylxanthine). Specific PDE inhibitors include PDEl, PDE2, PDE3, PDE4 and PDE5 specific inhibitors. In some embodiments, the PDE inhibitor is a PDE5 inhibitor. Specific PDE inhibitors contemplated herein include but are not limited Vinpocetine, EHNA (erythro-9-(2-hydroxy-3-nonyl)adenine), Anagrelide, Enoximine, Cilomilast, Etazolate, Glaucine, Ibudilast, Mesembrine, Rolipram, Pentoxifylline, Piclamilast, Dipyridamole, Acetildenafil, Avanafil, Sildenafil, Tadalafil, Udenafil, Vardenafil, Milrinone and Amrinone. The administration of combinations of PDE inhibitors is also contemplated.

An "effective amount" of a PDE inhibitor is the amount necessary or sufficient to have a medically desired biological effect in a subject (e.g., to prevent, reduce or reverse deleterious effects on blood vessels). Alternatively, the desired biological effect may include stimulating the growth of blood vessels. The effective amount will vary with the particular condition being treated, the age and physical condition of the subject being treated, the severity of the condition, the duration of the treatment, the nature of the concurrent therapy (if any), the specific route of administration and the like factors within the knowledge and expertise of the health care practitioner. This amount can be determined empirically using known methods and will vary from subject-to-subject. Generally, doses of PDE inhibitors would be from about 1 mg per day to 1000 mg per day. In some embodiments, the doses would be from 5 mg per day to 500 mg per day. In some preferred embodiments, the doses would be from 10 mg per day to 200 mg per day. Lower doses will result from other forms of administration, such as intravenous administration. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that subject's tolerance permits. In some embodiments, the dose of PDE inhibitor to be administered according to the methods of the disclosure is lower then the effective dose of PDE administered for the treatment of erectile dysfunction, pulmonary hypertension, high altitude pulmonary edema, asthma, stroke, cerebrovascular disorder (e.g., stroke), and congestive heart failure.

In the present methods, a PDE inhibitor is administered for an extended period of time. By "extended period of time" it is meant greater than 1 week, greater than 1 month, greater than 3 months, greater than 6 months, greater than 12 months, greater than 18 months, greater than 2 years, greater than 3 years, greater than 5 years, greater than 10 years, or more. Preferably, the PDE inhibitor(s) is administered continuously and substantially without interruption. In some embodiments, PDE inhibitor(s) is administered for a sufficient duration to have the desired biological effect in a subject (e.g., to prevent, reduce or reverse deleterious effects on blood vessels. Alternatively, the desired biological effect may include stimulating the growth of blood vessels).

The PDE inhibitor(s) may be administered by a variety of treatment schedules, such as on an hourly basis, on a daily basis, several times a day, several times a week, weekly, or monthly basis. The dose of PDE inhibitor(s) given will vary depending on the administration schedule.

In some embodiments of the disclosure the administration of PDE inhibitors is combined with methods for increasing nitric oxide levels in the subject. In some embodiments of the disclosure the administration of PDE inhibitors is combined with increased aerobic exercise. The "effective amount" and "extended period of time" of administration of the PDE inhibitor may be determined in conjunction with the aerobic exercise regimen. For instance, the effective amount of PDE inhibitor may be lowered when a more intense exercise regimen is followed and, vice versa, the effective amount of PDE inhibitor may be increased when the intensity of the exercise regimen is lowered.

In some embodiments, the administration of PDE inhibitors is combined with aerobic exercise. In some embodiments, the administration of PDE inhibitors is combined with additional therapeutic agents. In some embodiments, the administration of PDE inhibitors is combined with aerobic exercise and additional therapeutic agents. Additional therapeutic agents specifically include the nitric oxide inducers disclosed herein. Additional therapeutic agents, further include, but are not limited to, therapeutic agents used in the treatment of cardiovascular diseases. Examples of other therapeutic agents include, but are not limited to anti platelet drugs (e.g., aspirin, clopidogrel), beta- blockers (e.g., metoprolol, carvedilol), ACE inhibitors (e.g., captopril, enalapril), vasodilators (e.g., sodium nitroprusside), diuretics (e.g., bumetanide, chlortalidone), angiotensin II receptor antagonists (e.g., candesartan, eprosartan) and aldsterone antagonists (e.g., eplerenone, spironolactone).

The PDE inhibitor(s) and other therapeutic agent(s) may be administered simultaneously or sequentially. When the other therapeutic agents are administered simultaneously they can be administered in the same or separate formulations, but are administered at the same time. The administration of the other therapeutic agents and the PDE inhibitor(s) may also be temporally separated, meaning that the therapeutic agents are administered at a different time, either before or after, the administration of the PDE inhibitor(s). The separation in time between the administration of these compounds may be a matter of minutes or it may be longer.

When administered, the PDE inhibitor(s), and, optionally, other therapeutic agent(s) (e.g. a nitric oxide inducer) are preferably administered as pharmaceutical preparations applied in pharmaceutical ly-acceptable amounts and in pharmaceutically- acceptably compositions. Such preparations may contain salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutical ly-acceptable salts thereof and are not excluded from the scope of the disclosure. Such pharmacologically and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like. Also, pharmaceutically- acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.

The PDE inhibitor(s) and, optionally, other therapeutic agent(s) may be combined, optionally, with a pharmaceutically-acceptable carrier. The term "pharmaceutically-acceptable carrier" as used herein means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration into a human. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present disclosure, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.

The pharmaceutical compositions comprising the PDE inhibitor(s), and, optionally, other therapeutic agent(s), may contain suitable buffering agents, as described above, including: acetate, phosphate, citrate, glycine, borate, carbonate, bicarbonate, hydroxide (and other bases) and pharmaceutically acceptable salts of the foregoing compounds. The pharmaceutical compositions also may contain, optionally, suitable preservatives, such as: benzalkonium chloride, chlorobutanol, parabens and thimerosal.

The pharmaceutical compositions comprising the PDE inhibitor(s), and, optionally, other therapeutic agent(s), may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active agent into association with a carrier, which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.

The PDE inhibitor(s), and, optionally, other therapeutic agent(s), when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds (e.g., PDE inhibitor) may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.

Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active compounds (e.g., PDE inhibitor) may be in powder form for constitution with a suitable vehicle (e.g., saline, buffer, or sterile pyrogen-free water) before use.

Compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, pills, lozenges, each containing a predetermined amount of the active compound (e.g., PDE inhibitor). Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, elixir, an emulsion, or a gel.

Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, sorbitol or cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl- cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Optionally the oral formulations may also be formulated in saline or buffers, i.e., EDTA for neutralizing internal acid conditions or may be administered without any carriers.

Also contemplated are oral dosage forms of the above. A component (e.g., PDE inhibitor) or components may be chemically modified so that oral delivery of the derivative is efficacious. Generally, the chemical modification contemplated is the attachment of at least one moiety to the component molecule itself, where said moiety permits (a) inhibition of proteolysis; and (b) uptake into the blood stream from the stomach or intestine. Also desired is the increase in overall stability of the component or components and increase in circulation time in the body. Examples of such moieties include: polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline. Abuchowski and Davis, 1981, "Soluble Polymer-Enzyme Adducts" In: Enzymes as Drugs, Hocenberg and Roberts, eds., Wiley-Interscience, New York, NY, pp. 367-383; Newmark, et al., 1982, J. Appl. Biochem. 4: 185-189. Other polymers that could be used are poly-l,3-dioxolane and poly- 1,3,6- tioxocane. Preferred for pharmaceutical usage, as indicated above, are polyethylene glycol moieties.

For the component (or derivative) the location of release may be the stomach, the small intestine (the duodenum, the jejunum, or the ileum), or the large intestine. One skilled in the art has available formulations which will not dissolve in the stomach, yet will release the material in the duodenum or elsewhere in the intestine. Preferably, the release will avoid the deleterious effects of the stomach environment, either by protection of PDE inhibitor or by release of the biologically active material beyond the stomach environment, such as in the intestine.

To ensure full gastric resistance a coating impermeable to at least pH 5.0 is essential. Examples of the more common inert ingredients that are used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. These coatings may be used as mixed films.

A coating or mixture of coatings can also be used on tablets, which are not intended for protection against the stomach. This can include sugar coatings, or coatings which make the tablet easier to swallow. Capsules may consist of a hard shell (such as gelatin) for delivery of dry therapeutic i.e. powder; for liquid forms, a soft gelatin shell may be used. The shell material of cachets could be thick starch or other edible paper. For pills, lozenges, molded tablets or tablet triturates, moist massing techniques can be used.

The therapeutic (e.g., PDE inhibitor) can be included in the formulation as fine multiparticulates in the form of granules or pellets of particle size about 1 mm. The formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets. The therapeutic could be prepared by compression.

Colorants and flavoring agents may all be included. For example, the therapeutic {e.g., PDE inhibitor) may be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents.

One may dilute or increase the volume of the therapeutic (e.g., PDE inhibitor) with an inert material. These diluents could include carbohydrates, especially mannitol, lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch. Certain inorganic salts may be also be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.

Disintegrants may be included in the formulation of the therapeutic (e.g., PDE inhibitor) into a solid dosage form. Materials used as disintegrants include but are not limited to starch, including the commercial disintegrant based on starch, Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite may all be used. Another form of the disintegrants are the insoluble cationic exchange resins. Powdered gums may be used as disintegrants and as binders and these can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.

Binders may be used to hold the therapeutic (e.g., PDE inhibitor) together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both be used in alcoholic solutions to granulate the therapeutic.

An anti-frictional agent may be included in the formulation of the therapeutic (e.g., PDE inhibitor) to prevent sticking during the formulation process. Lubricants may be used as a layer between the therapeutic and the die wall, and these can include but are not limited to; stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000.

Glidants that might improve the flow properties of the therapeutic (e.g., PDE inhibitor) during formulation and to aid rearrangement during compression might be added. The glidants may include starch, talc, pyrogenic silica and hydrated silicoaluminate. To aid dissolution of the therapeutic (e.g., PDE inhibitor) into the aqueous environment a surfactant might be added as a wetting agent. Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents might be used and could include benzalkonium chloride or benzethomium chloride. The list of potential non-ionic detergents that could be included in the formulation as surfactants are lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation of the PDE inhibitor(s) either alone or as a mixture in different ratios.

Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.

Microspheres formulated for oral administration may also be used. Such microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the therapeutic (e.g., PDE inhibitor) for use according to the present disclosure may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,

dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. Also contemplated herein is pulmonary delivery of the therapeutic (e.g., PDE inhibitor). The therapeutic (e.g., PDE inhibitor) is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream. Other reports of inhaled molecules include Adjei et al., 1990, Pharmaceutical Research, 7:565- 569; Adjei et al., 1990, International Journal of Pharmaceutics, 63: 135-144 (leuprolide acetate); Braquet et al., 1989, Journal of Cardiovascular Pharmacology, 13(suppl. 5): 143- 146 (endothelin-1); Hubbard et al., 1989, Annals of Internal Medicine, Vol. Ill, pp. 206-212 (a 1- antitrypsin); Smith et al., 1989, J. Clin. Invest. 84: 1 145-1 146 (a- 1 -proteinase); Oswein et al., 1990, "Aerosol ization of Proteins", Proceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colorado, March, (recombinant human growth hormone); Debs et al., 1988, J. Immunol. 140:3482-3488 (interferon^/ and tumor necrosis factor alpha) and Platz et al., U.S. Patent No. 5,284,656 (granulocyte colony stimulating factor). A method and composition for pulmonary delivery of drugs for systemic effect is described in U.S. Patent No. 5,451,569, issued September 19, 1995 to Wong et al.

Contemplated for use in the practice of this disclosure are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art.

Some specific examples of commercially available devices suitable for the practice of this disclosure are the Ultravent nebulizer, manufactured by Mallinckrodt, Inc.,

St. Louis, Missouri; the Acorn II nebulizer, manufactured by Marquest Medical Products, Englewood, Colorado; the Ventolin metered dose inhaler, manufactured by Glaxo Inc., Research Triangle Park, North Carolina; and the Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford, Massachusetts.

All such devices require the use of formulations suitable for the dispensing of the therapeutic (e.g., PDE inhibitor). Typically, each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to the usual diluents, adjuvants and/or carriers useful in therapy. Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated. Chemically modified PDE inhibitors may also be prepared in different formulations depending on the type of chemical modification or the type of device employed. Formulations suitable for use with a nebulizer, either jet or ultrasonic, will typically comprise a PDE inhibitor(s) dissolved in water or other pharmaceutically acceptable solvent. The formulation may also include a buffer and a simple sugar (e.g., for stabilization of the PDE inhibitor(s) and regulation of osmotic pressure). The nebulizer formulation may also contain a surfactant, to reduce or prevent surface induced aggregation of the PDE inhibitor(s) caused by atomization of the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device will generally comprise a finely divided powder containing the PDE inhibitor(s) suspended in a propellant with the aid of a surfactant. The propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane,

dichlorotetrafluoroethanol, and 1,1 ,1 ,2-tetrafluoroethane, or combinations thereof. Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a surfactant.

Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder containing the PDE inhibitor(s) and may also include a bulking agent, such as lactose, sorbitol, sucrose, or mannitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation. The PDE inhibitor(s) should most advantageously be prepared in particulate form with an average particle size of less than 10 mm (or microns), most preferably 0.5 to 5 mm, for most effective delivery to the distal lung.

Nasal (or intranasal) delivery of a pharmaceutical composition of the present disclosure is also contemplated. Nasal delivery allows the passage of a pharmaceutical composition of the present disclosure to the blood stream directly after administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung. Formulations for nasal delivery include those with dextran or cyclodextran.

For nasal administration, a useful device is a small, hard bottle to which a metered dose sprayer is attached. In one embodiment, the metered dose is delivered by drawing the pharmaceutical composition of the present disclosure solution into a chamber of defined volume, which chamber has an aperture dimensioned to aerosolize and aerosol formulation by forming a spray when a liquid in the chamber is compressed. The chamber is compressed to administer the pharmaceutical composition of the present disclosure. In a specific embodiment, the chamber is a piston arrangement. Such devices are commercially available.

Alternatively, a plastic squeeze bottle with an aperture or opening dimensioned to aerosolize an aerosol formulation by forming a spray when squeezed is used. The opening is usually found in the top of the bottle, and the top is generally tapered to partially fit in the nasal passages for efficient administration of the aerosol formulation. Preferably, the nasal inhaler will provide a metered amount of the aerosol formulation, for administration of a measured dose of the drug.

The therapeutic (e.g., PDE inhibitor) may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the therapeutic (e.g., PDE inhibitor) may also be formulated as a depot preparation. Such long acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for inhalation, microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes, nebulized, aerosols, pellets for implantation into the skin, or dried onto a sharp object to be scratched into the skin. The pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above. The pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of methods for drug delivery, see Langer, Science 249: 1527-1533, 1990, which is incorporated herein by reference. The therapeutic agent(s), including but not limited to the PDE inhibitor(s), may be provided in particles. Particles as used herein means nano or micro particles (or in some instances larger) which can consist in whole or in part of the PDE inhibitor(s) or the other therapeutic agent(s) as described herein. The particles may contain the therapeutic agent(s) in a core surrounded by a coating, including, but not limited to, an enteric coating. The therapeutic agent(s) also may be dispersed throughout the particles. The therapeutic agent(s) also may be adsorbed into the particles. The particles may be of any order release kinetics, including zero order release, first order release, second order release, delayed release, sustained release, immediate release, and any combination thereof, etc. The particle may include, in addition to the therapeutic agent(s), any of those materials routinely used in the art of pharmacy and medicine, including, but not limited to, erodible, nonerodible, biodegradable, or nonbiodegradable material or combinations thereof. The particles may be microcapsules which contain the PDE inhibitor(s) in a solution or in a semi-solid state. The particles may be of virtually any shape.

Both non-biodegradable and biodegradable polymeric materials can be used in the manufacture of particles for delivering the therapeutic agent(s). Such polymers may be natural or synthetic polymers. The polymer is selected based on the period of time over which release is desired. Bioadhesive polymers of particular interest include bioerodible hydrogels described by H. S. Sawhney, CP. Pathak and J.A. Hubell in Macromolecules, (1993) 26:581-587, the teachings of which are incorporated herein. These include polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and

poly(octadecyl acrylate).

The therapeutic agent(s) may be contained in controlled release systems. The term "controlled release" is intended to refer to any drug-containing formulation in which the manner and profile of drug release from the formulation are controlled. This refers to immediate as well as non-immediate release formulations, with non-immediate release formulations including but not limited to sustained release and delayed release formulations. The term "sustained release" (also referred to as "extended release") is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that preferably, although not necessarily, results in substantially constant blood levels of a drug over an extended time period. The term "delayed release" is used in its conventional sense to refer to a drug formulation in which there is a time delay between administration of the formulation and the release of the drug therefrom. "Delayed release" may or may not involve gradual release of drug over an extended period of time, and thus may or may not be "sustained release."

Use of a long-term sustained release implant may be particularly suitable for treatment of chronic conditions. "Long-term" release, as used herein, means that the implant is constructed and arranged to deliver therapeutic levels of the active ingredient for at least 7 days, and preferably 30-60 days. Long-term sustained release implants are well-known to those of ordinary skill in the art and include some of the release systems described above.

For topical administration to the eye, nasal membranes, mucous membranes or to the skin, the PDE inhibitor(s) may be formulated as ointments, creams or lotions, or as a transdermal patch or intraocular insert or iontophoresis. For example, ointments and creams can be formulated with an aqueous or oily base alone or together with suitable thickening and/or gelling agents. Lotions can be formulated with an aqueous or oily base and, typically, further include one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents. (See, e.g., U.S. 5,563,153, entitled "Sterile Topical Anesthetic Gel", issued to Mueller, D., et al., for a description of a pharmaceutically acceptable gel-based topical carrier.)

In general, the PDE inhibitor(s) is present in a topical formulation in an amount ranging from about 0.01% to about 30.0% by weight, based upon the total weight of the composition. Preferably, the PDE inhibitor(s) is present in an amount ranging from about 0.5 to about 30% by weight and, most preferably, the PDE inhibitor(s) is present in an amount ranging from about 0.5 to about 10% by weight. In one embodiment, the compositions of the disclosure comprise a gel mixture to maximize contact with the surface of the localized pain and minimize the volume and dosage necessary to alleviate the localized pain. GELFOAM ® (a methylcellulose-based gel manufactured by Upjohn Corporation) is a preferred pharmaceutically acceptable topical carrier. Other pharmaceutically acceptable carriers include iontophoresis for transdermal drug delivery.

The disclosure also contemplates the use of kits. In some aspects of the disclosure, the kit can include a pharmaceutical preparation vial, a pharmaceutical preparation diluent vial, and PDE inhibitor(s). The vial containing the diluent for the pharmaceutical preparation is optional. The diluent vial contains a diluent such as physiological saline for diluting what could be a concentrated solution or lyophilized powder of the PDE inhibitor(s) . The instructions can include instructions for mixing a particular amount of the diluent with a particular amount of the concentrated

pharmaceutical preparation, whereby a final formulation for injection or infusion is prepared. The instructions may include instructions for treating a subject with an effective amount of the PDE inhibitor(s). It also will be understood that the containers containing the preparations, whether the container is a bottle, a vial with a septum, an ampoule with a septum, an infusion bag, and the like, can contain indicia such as conventional markings which change color when the preparation has been autoclaved or otherwise sterilized.

This disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including," "comprising," or "having," "containing," "involving," and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

Having thus described several aspects of at least one embodiment of this disclosure, it is to be appreciated various alterations, modifications, and improvements will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of this disclosure, and are intended to be within the spirit and scope of the disclosure. Accordingly, the foregoing description and drawings are by way of example only. The present disclosure is further illustrated by the following Example, which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference.

EXAMPLE

To determine the age-related effects in collaterogenesis, young (4 months) and aging (14 months) C57B1/6 mice were subjected to femoral artery ligation and extirpation and blood flow recovery was measured by Laser-Doppler flow. Compared to the young 4 month old mice, collateral flow recovery was markedly impaired in 14- month-old mice (Figure IA). In addition, the necrotic score is greater in the ischemic calf in 14 vs. 3 month-old mice

To define some of the mechanisms responsible for the age-related impaired collateral flow recovery response that occurs with aging, a series of studies to determine: a) some of the molecular abnormalities that develop with aging that could be causally relating to impaired collaterogenesis; and

b) any collateral phenotype changes that could contribute to the abnormal flow recovery were performed.

Expression of molecules normally up-regulated in response to tissue ischemia or injury was measured in aging vs. young mice. It was reasoned that if the particular collaterogenic contribution of a given molecule were not impaired with aging, the expression of such a molecule in response to ischemia would be up-regulated to a greater extent in old vs. young mice (because of the greater ischemia). If, however, aging resulted in a paradoxical decrease in expression, this would suggest that such a molecule and its signaling pathways might be contributing to the impaired collaterogenic response found with aging.

Figure 2 demonstrates that MMP9 mRNA (and protein), SDF- lα mRNA and HIF- lα mRNA levels were significantly higher in aging vs. young mice on day 7 post ischemia induction. Although not wishing to be bound by any particular theory, the most reasonable interpretation of the ischemia-responsive genes that were expressed more robustly in older vs. younger mice is that their greater expression reflects the more severe ischemic stimulus experienced by the older mice and that there is no important impairment in gene expression.

Figure 3 shows the expression profile of two genes that exhibit decreased expression in aging mice despite the great ischemic insult. There is an early increase in vascular endothelial growth factor (VEGF) expression in the young mice in the first 2 hours following femoral artery ligation— changes not seen in the old mice. Despite greater ischemia in older mice, by day 7 there are lower levels of VEGF mRNA and protein present in the ischemic calf of the old vs. young mice. Panel C: endothelial nitric oxide synthase (eNOS) protein levels are reduced in old mice on both day 3 and 7. Of interest, the older mice also exhibit lower baseline levels of eNOS protein. These results indicate that aging impairs the ability to increase VEGF expression in response to ischemia, reduces the levels of eNOS present following ischemia induction, and also lowers baseline eNOS protein levels. These changes are in the direction opposite to that which would be expected, in view of the more severe ischemia present in older mice. Thus, the data indicate that impaired VEGF expression and reduced eNOS levels may contribute to the impaired collateral flow recovery demonstrated by the older mice.

Changes in CD26 mRNA expression in bone marrow were measured in young vs. aging mice before and after femoral artery ligation and extirpation (Figure 4). Amongst CD 26's activities is its ability to degrade SDF-I, a molecule that binds progenitor cells expressing CXCR4 to bone marrow stroma. By eliminating the SDF-1/CXCR4 bond, CD26 participates in mobilization of progenitor cells; its decrease suggests impaired mobilization capacity. Panel A demonstrates that young mice exhibit increased expression of CD26 under control (baseline) conditions, a difference that remains during hindlimb ischemia. Panel B demonstrates that differences in mRNA are also reflected in differences in the number of cells expressing CD26 protein.

When bone marrow (BM) obtained from aging mice was transplanted into young mice, mobilization and homing were impaired (Figure 5). Young mice had good collateral flow recovery regardless of age of BM donor. Remarkably, however, although transplantation of young BM into old mice increased homing of BM cells to ischemic tissue, the greater homing did not improve the impaired flow recovery of the old mice. Even when old mice received BM transplant from young mice, there was no

improvement in flow, indicating that mobilization of BM cells and homing (even though the BM cells are derived from young mice) are not the dominant effect of the aging- related impairment in collaterogenesis.

Using a Matrigel plug angiogenesis assay, it was found that the capacity for neovascularization to occur in response to angiogenic stimuli was impaired with aging (Figure 6). Young (7-9 wks, n = 14) and old (22 months n= 14) male C57 Bl/6 mice were subcutaneously injected with matrigel infused with either bFGF, at a concentration of lmg/ml or 5mg/ml, or PBS (control). After 5 days, the plugs were harvested and stained using H&E. Angiogenesis was assessed as endothelial invasion of the matrigel plug. Results were analyzed using a two-way ANOVA. Angiogenesis was markedly impaired with aging, as fewer endothelial cells were present in matrigel plugs at all concentrations of bFGF (p= 0.0004). Thus, reduced target-tissue responsiveness to collaterogenic stimuli contributes to aging-related impaired collaterogenesis.

Next, brain collateral phenotype changes in young vs. aging mice were measured. It was found that in the young mouse, collaterals are typically straight while in the old mouse, they are often tortuous (Figure 7, panels A and B).

In addition, six randomly chosen collaterals were measured in each animal.

Tortuosity index is calculated as axial length divided by scalar length. Since tortuosity increases resistance to flow, the increased tortuosity of collaterals present in old mice increases collateral resistance (Figure 7C). The aging-related decrease in collateral diameter was found to be 13% (Figure 7D) and the changes in tortuosity and diameter led to an increase of 139% in collateral resistance (Figure 7E). In contrast, the number of collaterals did not reduce with age. Values were obtained by counting all the collaterals interconnecting the anterior and middle cerebral arteries of both hemispheres, after fixing the vasculature at maximal diameter induced by papavarine and adenosine and filling with a casting agent. Arteriogram of 3 month old group obtained with FITC-albumin barium-gel. 16-month group filled with PU4ii polyurethane. The two casting materials give identical morphometric measures.

These results indicate that in aging mice (14-16 months of age) there are marked changes in collaterals, and that these changes are systemic in nature, involving at least the hindlimb and cerebral collaterals. In terms of the cerebral collaterals, although by 14-16 months of age there is no vessel dropout, there are changes in diameter and tortuosity that lead to substantial increases in calculated collateral resistance. Thus, if a stroke occurs under these conditions, (i.e., occlusion of the middle cerebral artery) the older mice have less than half the collateral flow to the dependent cortex, a change predisposing to a more severe infarction.

Despite the changes produced with aging in the cerebral vessels of 14-16 month- old mice, no differences in hindlimb flow immediately post femoral artery ligation were observed (Figure 1 ; Figure 8, in which high sensitivity laser-Doppler flow was used). Thus, either no such changes are present in the hindlimb of 14-16 month-old mice, or that the changes are sufficiently small to avoid detection in flow changes with current methodology.

However, there are two prominent flow changes present in the aging 14-16 month-old mice: a) a decrease in flow recovery, and b) a decrease in the rate of increase in collateral flow recovery over the first few days post ligation. This latter effect is seen in both female mice with aggressive hindlimb surgery (femoral artery ligation and extirpation-Figure 1) as well as in male mice with simple femoral artery ligation (Figure 8). These data indicate that no matter what changes occur in collateral number in the hindlimb by this age, it does not result in differences in flow immediately following femoral artery ligation (i.e., no measurable differences in the immediate function of preexisting collaterals). However, there is a marked aging-related decrease in the rate of flow recovery, a finding indicating a defect in collateral positive remodeling (or, impaired neoangiogenesis). Although not wishing to be bound by any particular theory, this change importantly contributes to the overall defect in functional flow recovery.

Figure 9 demonstrates that just as there is increased tortuosity of the pial collaterals, there is also increased tortuosity of the hindlimb collaterals (arrows point to tortuous collaterals in the old mouse). Figures 9A-C show typical micro-CT images of hindlimb collaterals in (A) young mouse thigh, (B) older mouse thigh, (C) eNOS KO mouse thigh. Micro-CT assessment (16μm resolution) of preexisting collaterals was performed immediately following surgery. All arteries possessing at least one layer of smooth muscle cells (therefore considered arteries) and coursing through the thigh and calf were counted. Analysis was then carried out by batching the groups into vessels <24 μm and >24μm. In the calf, both older and eNOS KO mice had fewer vessels <24μm than young mice; in the thigh, there were significantly fewer such vessels present in the older mice, with a similar trend in the eNOS KO mice (Fig 9D). For vessels >24μm, there were fewer vessels present in both old and eNOS KO mice vs. young mice in the calf, although there were no differences among the groups in the thigh (Fig 9E).

In this same group of aging mice (19-24 month-old female C57/B16 mice), the number of small arteries (collaterals) in the calf and in the hindlimb were also measured. As these mice become very old there is a dropout of small arteries (<24μM in diameter) both in the thigh and in the calf (Figure 10). Without wishing to be bound by any particular theory, this arterial/collateral pruning may profoundly reduce not only the capacity of collaterals to supply blood to distal tissues upon acute obstruction of the major native feeder artery, but also the capacity of the collaterals to help the limb recover function chronically. The artery dropout may have important implications relating to the potential efficacy of therapeutic interventions. It raises one important question of whether, once the number of collaterals decrease as a result of the dropout process, therapeutic interventions would be effective—or will it be necessary to intervene therapeutically before collateral dropout, with treatment strategy primarily targeted to preventing dropout.

Given the decrease in eNOS expression found in aging mice (Figure 3), the effects on collateral development in the absence of eNOS expression in female eNOS knock out (KO) mice were examined. Micro-CT studies immediately after femoral artery ligation and extirpation revealed that the eNOS KO mice exhibited a decrease in the number of small arteries (<24μM in diameter) when compared to comparatively aged wild type mice (3-4 months of age). These KO mice had a similar reduction in the number of vessels as the aged mice. (Figure 1 1- all mice were operated upon and analyzed as a single study). We were intrigued by the finding that baseline levels of eNOS expression, independent of ischemia, were decreased with aging (Figure 12). This was the only gene of the group we studied, including VEGF (Fig. 3B), whose baseline expression

(independent of ischemia) was impaired by aging.

In addition to the aging-induced decreased baseline levels of eNOS protein, we determined whether aging also impaired eNOS signaling. Because phosphorylation of eNOS at serine 1 177 (ser-1 177) is critical for eNOS signaling and for NO generation, we determined whether phosphorylation at this site was reduced by aging. We found this to be the case (Figure 12A). Importantly, the ratio of phosphorylated eNOS to total eNOS was also significantly lower in the older vs. young mice (p=0.015), indicating that decreased phosphorylated eNOS protein, and therefore decreased eNOS signaling, was due not only to decreased eNOS protein but also to an impaired mechanisms involved in ser-1 177 phosphorylation (Figure 12B).

Given the central role eNOS/NO plays in vascular function, including a probable role in preventing aging-related endothelial cell apoptosis, we hypothesized that the detrimental effects aging had on eNOS and eNOS signaling might compromise the integrity of the vascular system. We tested this hypothesis by using the high resolution capacity of micro-CT to analyze all arteries from the level of micro-CT resolution (16μm) in the thigh and calf. Our findings confirmed the hypothesis: aging was indeed associated with a loss of arterioles. To confirm a causal role of eNOS in the aging- related dropout of microvessels, we performed similar studies in young eNOS KO mice. We found such mice have fewer pre-existing collaterals in their hindlimb when compared to young wild type mice - the same pattern observed in old wild type mice (Figure 9). These findings strongly suggest that impairment of the NO pathway may contribute to the aging-induced loss of collaterals. Interestingly, we found a similar pattern of aging-related collateral dropout in the brain (Figure 7).

Aging causes deleterious phenotypic and functional changes in pre-existing native collaterals in the hindlimb, including collateral dropout.

To elucidate mechanisms responsible for compromised collateral flow recovery associated with risk factors, prior studies focused on possible reduced collateral remodeling following ischemia. However, because native collaterals are present at birth, we hypothesized that aging causes deleterious phenotypic and functional changes in these pre-existing collaterals before obstructive disease develops. Our studies confirmed this hypothesis: aging caused a decline in diameter and an actual dropout (rarefaction) of native collaterals; positive remodeling was also impaired after occlusion of the conduit artery (Fig. 13). This microvascular rarefaction, decrease in collateral diameter, and impaired positive remodeling following acute femoral artery ligation were accompanied by greater functional compromise of the ischemic hindlimb (decreased motor function and atrophy) in older vs. younger mice.

Aging-induced collateral changes in phenotype and function occur not only in the hindlimb, but also in the brain-- including collateral dropout.

Fig 14a demonstrates that the aging-induced deleterious changes in native collateral number, diameter and length (tortuosity) increased calculated collateral resistance (by ~ 8-fold!). In addition, Fig. 14b demonstrates that aging also compromises collateral remodeling induced by occlusion of the conduit artery— in this case the middle cerebral artery (MCA). Consistent with these deleterious collateral changes, MCA occlusion was associated with a 3-fold increase in infarct volume in old vs. young mice (Fig. 14c).

Aging-induced abnormalities in eNOS/NO pathway contribute to collateral dropout.

Our attention was directed to a possible role of the eNOS/NO system in aging- induced collateral rarefaction since aging is associated with reduced functionality of eNOS/NO8. In an initial study we determined the effects of aging on levels of eNOS protein and activation of eNOS (phosphorylation of serine 1 177). We found (Fig. 15) that eNOS protein is decreased in the calf of old mice, and that its activation (as determined by phosphorylation of serine 1 177) is markedly impaired, whether normalized for GAPDH or eNOS.

Young eNOS knockout mice, like old wild-type mice, exhibit microvascular dropout.

To determine if the association between decreased eNOS protein and activation observed in old mice (Fig 15) reflected causality, we examined vessel phenotype in eNOS knockout (KO) mice (Fig 9). Immediately after femoral artery occlusion the number of small arteries was less in young eNOS KO mice vs. young wildtype mice— a similar reduction as the aged wildtype mice. The collateral rarefaction we observed in eNOS KO mice did not result from decreased collateral number in utero, as we have found that eNOS KO mice have normal collateral number at birth, but exhibit rapid collateral dropout over the first 3 months of life. These data provide strong evidence indicating that dysfunction of the eNOS/NO system is a significant contributor to collateral dropout in aging. We then examined how a dysfunctional eNOS/NO system might contribute mechanistically to collateral dropout.

Endothelial cells derived from old vs. young mice are more sensitive to apoptosis, an effect appearing to be mediated, at least in part, by dysfunctional eNOS.

We found that, as in the calf (Fig 15) ECs obtained from older mice (24 vs. 3 months-old) exhibited decreased eNOS protein and serine-1 177 phosphorylation (data not shown). Because the eNOS/NO pathway exerts anti-apoptotic effects, we examined the sensitivity of these cells to an apoptotic stimulus (TNFα). The results are shown in Fig 16. The fact that ECs are rescued from an apoptotic stimulus by an NO donor such that the magnitude of apoptosis exhibited by cells derived from old mice is no different from that of cells derived from young mice—suggests dysfunctional eNOS signaling contributes to the aging-related increased sensitivity to apoptosis.

SMCs derived from old mice are also more sensitive to an apoptotic stimulus, an effect attenuated by tadalafil, a PDE5 inhibitor.

Another potential mechanism by which aging causes collateral dropout was suggested by our preliminary finding (Fig 17) that vascular SMCs also exhibited increased apoptotic sensitivity to H₂O₂ (an apoptotic stimulus, that works through a pathway different from TNFα). Since eNOS/NO is not a major primary signaling pathway in SMCs— instead affecting SMCs through NO secreted by ECs that is taken up by the SMC (Fig 20), the increased in vitro sensitivity of SMCs to apoptosis (i.e., in the absence of ECs) implies another causal mechanism, such as aged-induced increased oxidative stress. We have tested PDE5-I as a strategy to prevent aging-induced collateral dropout. To test whether PDE5-I has a broader range of activity on age-related changes than just decreasing cGMP degradation, we investigated the effect of Tadalafil (Cialis) on the increased propensity of SMCs to undergo apoptosis. Tadalafil inhibited H2O2 - induced apoptosis in SMCs derived from old mice (Fig 17, right panel).

These data presented in Figures 16 and 17 have important mechanistic implications. If aging increases apoptotic propensity of ECs and SMCs, the integrity of vessels they line will be compromised. This could partly explain the aging-related collateral dropout (and perhaps also the decreased collateral diameter). This implies that PDE5-I could exert protective effects through 2 dominant mechanisms: 1) enhancement of the eNOS/NO signaling pathway; 2) reduction of increased oxidative state induced by aging.

P-VASP, a downstream molecule formed by activation of an intact eNOS/NO/cGMP system (and therefore a marker of intact eNOS/NO/cGMP signaling), is increased by exercise.

To test the integrity of the eNOS/NO/cGMP pathway in vivo, we performed preliminary studies to determine a) if we could develop a technique that would allow us to quantitatively measure P-VASP in an artery, b) whether the chronic exercise

(swimming) we plan to use is feasible, and c) if such exercise enhances the

eNOS/NO/cGMP pathway (reflected by increased P-VASP). We succeeded in measuring P-VASP by western blot, and found data suggesting that the chronic exercise we will employ enhances eNOS/NO/cGMP pathway activity (Fig 18).

PDE5 inhibition with tadalafil enhances collateral function follow ing femoral artery occlusion.

To test the feasibility that even relatively short-term PDE5-I in old mice enhances collateral function on a background of chronic exercise, we determined whether collateral flow in 22 months old mice is augmented by 12 days of tadalafil (Fig 19). Tadalafil significantly increased collateral flow. This adds important insights as to possible multiple mechanisms by which aging impairs and PDE5-I improves collateral function, as this short duration Rx could not have decreased collateral dropout. The data therefore suggest that in old mice there still is an element of reversible collateral dysfunction (decreased diameter, increased tortuosity, and decreased positive remodeling), such that although PDE5-I cannot restore lost collaterals, it still can cause, presumably thru relaxed SMC tone and thus vasodilation, or by collateral restructuring, changes contributing to and augmenting the effects we expect from more prolonged treatment, per Aim 3, especially when begun before advancing age causes significant collateral dropout.

Development of immunohistochemical assays for assessment and cellular localization of age-induced changes in eNOS/NO signaling in collaterals.

Since collateral vessels in mice are small and difficult to dissect free of surrounding tissue, we have developed and validated immunohistochemical assays for several of the key molecules we wish to localize to either ECs or SMCs of collaterals.

Immunohistochemistry (DAB/hematoxylin counterstain) of semimembranosus muscle collaterals are stained for nitrotyrosine (an oxidation product of peroxy nitrite, a product of uncoupled eNOS), phosphorylated eNOS (an activated form of eNOS), and phosphorylated VASP (a readout of normal eNOS signaling) in mice. Immunopositivity for p-eNOS is present primarily in ECs, which would be expected since the major localization of eNOS is in ECs; conversely, the major immunopositivity for p-VASP is in SMCs, where this molecule is formed by cGMP activity (which is increased by NO from ECs). Immunopositivity for nitrotyrosine is present in both EC and SMC cytoplasm, which is compatible with the concept that peroxy nitrite, produced by dysfunctional EC eNOS, diffuses to adjacent cells causing tyrosine nitrosylation.

These assays enable us to determine aging-related changes and the effects of PDE5 inhibition and of exercise in collaterals, and in which cell type.

Changes in P-VASP occurring within the collateral wall following conduit artery occlusion demonstrates aging-induced impaired eNOS signaling.

In our first series of collateral-specific experiments using this

immunohistochemical methodology, we identified collaterals present in the

semimembraneous muscles in the thigh of both the hindlimb undergoing FA ligation and the contralateral hindlimb (control) 7 days after FA occlusion. We then quantitatively analyzed, by immunohistochemical staining, changes in levels of P-VASP (vasodilator- stimulated phosphoprotein, phosphorylated at serine 239) in collaterals. Occlusion of the major conduit artery (which increases shear stress in the accompanying collaterals) was associated in old mice with critical evidence of an impaired eNOS/NO pathway— reduced P-VASP expression (Fig. 20). Interestingly, P-VASP actually decreased (panels B, C), suggesting eNOS uncoupling. Thus, aging markedly interferes with normal eNOS signaling in collaterals following acute occlusion of the major conduit artery, a change associated with, at best, impaired positive remodeling, at worst, actual negative remodeling.

Aging is associated with an increase in vascular oxidative stress.

Coupled eNOS dimer residing within endothelial cells synthesizes NO, which then is secreted and taken up by smooth muscles cells. NO stimulates formation of cGMP, which then activates cGMP-dependent protein kinase I (cGK-I), an enzyme that leads to multiple beneficial vascular effects including the generation of P-VASP. In contrast conditions can arise, such as those leading to increased oxidative stress, that lead to an uncoupling of eNOS and formation of eNOS monomer. Under this circumstance, when eNOS is activated (phosphorylated) superoxides and peroxynitrate are formed (rather than NO), thereby increasing oxidative stress that pre-disposes to multiple deleterious affects, including an increased propensity to apoptosis. Thus, an increase in phosphorylation of eNOS does not necessarily predict activation of a signaling cascade that would produce the beneficial effects usually associated with, and considered an intrinsic part of, eNOS signaling.

We therefore determined whether one of major conditions that can alter normal eNOS/NO signaling was present in our model of aging— increased vascular oxidative stress. We confirmed this to be the case (Fig. 21).

Aging is associated with increased expression ofPDES in SMCs.

We then focused on another critical component of the eNOS/NO pathway that could, if increased by aging, impair the beneficial effects of NO— PDE5 activity (Fig 22). As shown herein, aging increases PDE5 activity, which provides a new mechanism for aging-induced dysfunction of eNOS, and provides an additional mechanism whereby chronic administration of PDE inhibitors exert beneficial effects on collaterals as an individual ages. Therapeutic preventive strategy

A therapeutic preventive strategy is presented that attenuates the impairment of the eNOS/NO system that causes deleterious effects on collaterogenesis in aging. The strategy is based on the observations that (1) cGMP is a second messenger in the Nitric Oxide (NO) cascade activation; (2) phosphodiesterases (PDEs) degrade cGMP, thereby decreasing eNOS/NO induced activation of the target tissue (vasodilation of the arterial system); (3) and physical exercise increases NO release from vascular endothelium. Thus, the combination of the PDE5 inhibitors (which stabilizes cGMP levels) and repeated bouts of exercise (which increase vascular NO release and therefore increase cGMP), both initiated during "middle age" before microvascular dropout has occurred, work together to prevent the subsequent aging-related dropout of collateral vessels in the hindlimb, heart, and brain.

The strategy is confirmed by the following experiment: C57B16 mice are divided in six groups: 1) Control Young (Ctrl y) - young mice (4 months old) that are not be subjected to intervention; 2) Control 10-month-old (Ctrl 10) - 10-month-old mice that are not be subjected to intervention; 3) Control Old (Ctrl old) - old mice (22 months old) that are not be subjected to intervention; 4) Exercise - 10-month-old mice are subjected to swimming for 12 months, until 22 months of age; 5) PDE5 inhibitors - 10-month-old mice receive PDE5 inhibitors until 22 months of age; and 6) exercise and PDE5 inhibitors (Exerc + PDE5) - 10-month-old mice receive both treatments: exercising training and PDE5 inhibitors for a 12 month period. The details of the experimental setup are provided below:

C57B16 mice are divided in six groups:

1. Control Young (Ctrl y) - young mice (4 month-old) that are not subjected to

intervention;

2. Control Old (Ctrl old) - old mice (22 month-old) that are not subjected to intervention;

3. Control 10-month-old (Ctrl lOmo) 10-month-old mice that are not subjected to

intervention to show how are the phenotype and function of collaterals in these mice when they start the treatment with PDE5 inhibitors and exercise;

4. Exercise (Exerc) - 10-month-old mice are subjected to swimming training (sessions 3 times/week, each session of 45 minutes per 12 months) until 22 months of age; 5. PDE5 inhibitors - 10-month-old mice will receive PDE5 inhibitors

6. Exercise and PDE5 inhibitors (Exerc + PDE5 inhibitors) - 10-months-old mice

receiving both treatments: exercising training and PDE5 inhibitors for a 12 month period.

Each group includes 28 mice: 6 mice for Micro CT experiments; 10 mice for LPDI and Postmortem Pial Microangiography - these mice are used first for LPDI, showing their flow recovery until day 28, and after for Postmortem Pial

Microangiography since there is no changes in collaterals brain phenotype related with hindlimb ischemia; and 12 mice for Western Blot [6 mice per time point - baseline (pre- ligation) and day 3 (post ligation)].

In the long-term exercise groups the 10-month-old mice exercise by swimming in a tank with 58m in length, 42cm in width and 40cm in height. The plastic tank allows for observation of the mice even when they are under the water. The temperature of water is 30 - 32⁰C and the depth will be 30cm. The water is changed once a day, after each swimming section, and the tank is cleaned with regular soap. Mice swim for 3 days/week during 15 minutes in the first week and the swimming time is gradually increased in 10 minutes per week until achieve 45 minutes per session. The mice are monitored while they swim. Mice are towel-dried after each training session, and observed during the entire swimming session. The 10-month-old mice exercise for 12 months until 22 months of age.

The number of mice receiving PDE5 inhibitors is 56 mice (2 groups: PDE5 inhibitors and Exercise + PDE5 inhibitors).

At the end of the study, animals are sacrificed; adductor and calf muscle are collected for protein extraction, at baseline (pre-ligation) and day 3 (post ligation) (n=6 per time point). Micro CT studies are performed on a separate group of mice (n=6). A third group of mice is subjected to hindlimb ischemia and followed using LDPI, and an analysis of collateral diameter and tortuosity in the brain.

In the hindlimb ischemia model: mice are subjected to operative intervention to create unilateral hindlimb ischemia. Hair is removed from the surgical site and the site is prepared with betadine, chloexidine or similar disinfectant. All the animals are covered with a sterile surgical drape. Surgical instruments are sterilized using an autoclave. Instruments are sterilized using a bead sterilizer in between animals. Exposure of the femoral artery is obtained by performing an incision in the skin overlying the middle portion of the hind limb of each mouse. After carefully dissecting the femoral artery from the femoral vein and nerve, the femoral artery is ligated, at the level of the popliteal bifurcation. The overlying skin is closed with either a monofilament nonabsorbable suture, or a staple. During the procedure sterile instruments and sterile materials (gloves, instruments, drapes etc.) are used, as well as aseptic techniques. The surgeon's attire includes a mask, bouffant, and a lab coat. An operating microscope is used during the surgical procedure.

Laser Doppler Perfusion Imaging (LDPI) is used to record serial blood flow measurement preoperatively, immediately post-operatively, 3, 7, 14, 21 and 28 days after the surgery. Excessive hair is removed from the limb before imaging, and the mice is placed on a heating pad at 37⁰C to minimize temperature variation. Consecutive images are obtained over the region of interest (leg and foot). Each imaging procedure takes roughly 30 minutes to complete.

Western Blot is performed after protein extraction using antibodies against eNOS, phosphorylated eNOS, and VEGF. The GAPDH housekeeping gene is used as internal control. The mice are sacrificed and total protein is extracted form calf, thigh, and adductor muscles in baseline and in day 3. The target proteins are detected using specifics antibody for eNOS, eNOS phosphorylated (Ser 1177) and VEGF. The quantification of them are normalized by GADPH.

Postmortem pial microangiography is used to evaluate the collaterals phenotype. The thoracic aorta is cannulated, blood is cleared with PBS and the cerebral circulation is maximally dilated with papavarine-PBS containing Evans blue dye for 5 minutes from a pressure reservoir to allow extravasation of dye to enhance vascular contrast and mark infarction limits. A craniotomy is then rapidly performed and fixative applied topically to the cortex to fix dye diffusion and the vasculature at maximal dilation. After craniotomy, lead-chromate yellow latex Microfil (Flow Tech, Inc) is infused with the aid of a stereomicroscope to insure filling of all collaterals. The viscosity is formulated to prevent contrast from crossing capillaries. This prevents obscuring the collateral circulation by the filling of capillaries and venules/veins. Image analysis here and elsewhere is with ImageJ, Photoshop or Metamorph. All analyses are done by blinded observers.

In Microfil injection for Micro CT: 1) to prepare heparinized PBS: 20U/ml; Make 40ml, 0.8ml of stock of heparin in 40ml PBS. For heparinizing the mouse, 400U/kg, 0.4U/g, 8U/20g, inject 0.4ml of heparized PBS through inferior vena cava. 2) Lead/Latex Indicator (Microfil): Solution is mixed in a 1 :1 ratio (5ml Indicator + 5ml Diluent) in a conical tube. Hardener is added just prior to injection (500ul), stir for 5 minutes. Syringe is filled and all air removed before each injection. 3) Attach one end of Baxter extension tubing to a 3-way stopcock, attach another end to a 23G needle, and fill the tubing with heparinized PBS so there are no air bubbles. The procedures is: 1) after the operation of hindlimb ischemia, heparinzine the mouse by the injection of 0.4ml of heparinized PBS retro-orbitally for 10 minutes. 2) Open the pleural cavity. 3) Dissect the thoracic artery just above the diaphram, put two sutures under the artery, insert 24G AngioCath into the thoracic aortic artery, fix the catheter by the two sutures. 4) Perfuse the mouse body with 20ml of vasodilation buffer (e.g. PBS with papaverin 4 mg/1, adenosine 1 g/1, heparine 20U/ml) at the rate of 2ml/min. 5) Perfuse the mouse with 2% paraformaldehyde 10ml (in PBS) for 5 minutes. 6) Inject the microfil, about 0.2 mL/10 g body weight at 0.35 ml/min using a syringe pump (mechanical injector). 7) Leave the animal at 4C overnight. 8) Next day the skin on bilateral hindlimbs is removed. Remove bilateral hindlimbs and store them in a 50ml conical tubes containing 10% formalin. Perform micro-CT scan.

Equivalents

The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the disclosure. The present disclosure is not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect of the disclosure and other functionally equivalent embodiments are within the scope of the disclosure. Various modifications of the disclosure in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects of the disclosure are not necessarily encompassed by each embodiment of the disclosure.

Claims

CLAIMS What is claimed is:

1. A method of preventing, reducing or reversing one or more deleterious effects on a blood vessel in a subject, the method comprising:

administering to a subject in need thereof an effective amount of a

phosphodiesterase (PDE) inhibitor over an extended period of time to prevent, reduce or reverse one or more deleterious effects on a blood vessel in the subject.

2. The method of claim 1, further comprising increasing nitric oxide levels in the subject.

3. The method of claim 2, wherein the nitric oxide levels in the subject are increased through aerobic exercise.

4. The method of claim 2, wherein the nitric oxide levels in the subject are increased through the administration of a nitric oxide inducer.

5. The method of any one of claims 1-4, wherein the preventing, reducing or reversing one or more deleterious effects on a blood vessel comprises suppressing a loss of arterioles, suppressing a loss of collaterals, suppressing collateral dysfunction or suppressing a decrease in collateral flow recovery.

6. The method of any one of claims 1-5, wherein the subject is otherwise free of indications for treatment with the phosphodiesterase (PDE) inhibitor.

7. The method of any one of claims 1-6, wherein the deleterious effect is caused by aging, hypercholesterolemia, hypertension, hyperlipidemia, obesity, diabetes mellitus, smoking, genetic predisposition or lifestyle.

8. The method of any one of claims 1-7, wherein the blood vessel is present in at least one of the following: heart, brain, extremity, or kidney.

9. The method of any one of claims 1-8, wherein the subject is at risk of a cardiovascular condition.

10. The method of claim 9, wherein the cardiovascular condition is: heart attack, stroke, renal failure, claudication, loss of leg caused by aging,

hypercholesterolemia, hypertension, hyperlipidemia, obesity, diabetes mellitus, smoking, or genetic predisposition.

1 1. The method of any one of claims 1-10, wherein the phosphodiesterase (PDE) inhibitor is selected from the group consisting of Vinpocetine, EHNA (erythro-9- (2-hydroxy-3-nonyl)adenine), Cilomilast, Etazolate, Glaucine, Ibudilast, Mesembrine, Rolipram, Acetildenafil, Avanafil, Sildenafil, Tadalafil, Udenafil, Vardenafil, Milrinone and Amrinone.

12. A method for preserving or increasing the functional capacity of a blood vessel in a subject, the method comprising:

administering to a subject in need thereof an effective amount of a

phosphodiesterase (PDE) inhibitor over an extended period of time to preserve or increase the functional capacity of the blood vessel in the subject.

13. The method of claim 12, further comprising increasing nitric oxide levels in the subject.

14. The method of claim 13, wherein the nitric oxide levels in the subject are increased through aerobic exercise.

15. The method of any one of claims 12-14, wherein the subject is otherwise free of indications for treatment with the phosphodiesterase (PDE) inhibitor.

16. The method of any one of claims 12-15, wherein the blood vessel is present in at least one of the following: heart, brain, extremity or kidney.

17. The method of any one of claims 12-16, wherein the phosphodiesterase (PDE) inhibitor is selected from the group consisting of Vinpocetine, EHNA (erythro-9- (2-hydroxy-3-nonyl)adenine), Cilomilast, Etazolate, Glaucine, Ibudilast, Mesembrine, Rolipram, Acetildenafil, Avanafil, Sildenafil, Tadalafil, Udenafil, Vardenafil, Milrinone and Amrinone.

18. A method for reducing the incidence and/or severity of at least one of the following conditions: heart attack, stroke, renal failure, claudication, loss of limb or vascular occlusions, the method comprising:

administering to a subject in need thereof an effective amount of a

phosphodiesterase (PDE) inhibitor over an extended period of time to reduce the incidence and/or severity of at least one of the conditions selected from the group consisting of heart attack, stroke, renal failure, claudication, loss of limb and vascular occlusions.

19. The method of claim 18, further comprising increasing nitric oxide levels in the subject.

20. The method of claim 19, wherein the nitric oxide levels in the subject are increased through aerobic exercise.

21. The method of any one of claims 18-20, wherein the subject is otherwise free of indications for treatment with the phosphodiesterase (PDE) inhibitor.

22. The method of any one of claims 18-21, wherein the blood vessel is present in at least one of the following: heart, brain, extremity or kidney.

23. The method of any one of claims 18-22, wherein the phosphodiesterase (PDE) inhibitor is selected from the group consisting of Vinpocetine, EHNA (erythro-9- (2-hydroxy-3-nonyl)adenine), Cilomilast, Etazolate, Glaucine, Ibudilast, Mesembrine, Rolipram, Acetildenafil, Avanafil, Sildenafil, Tadalafil, Udenafil, Vardenafil, Milrinone and Amrinone.