WO2007049088A1

WO2007049088A1 - Method of stimulating growth of functional blood vessels and/or regeneration of myocardium in damaged tissues

Info

Publication number: WO2007049088A1
Application number: PCT/IB2005/003191
Authority: WO
Inventors: Ming Li; Lei Cheng; Xin-Sheng Yao; Hong-Wei Liu; John Elsby Sanderson; Xue-Mei Gu; Leonard Wing-Hong Cheung
Original assignee: Lead Billion Limited
Priority date: 2005-10-27
Filing date: 2005-10-27
Publication date: 2007-05-03
Also published as: WO2007049088A8

Abstract

Use of Gemin A from an extract of Geum Japonicum for the manufacture of a medicament for stimulating growth of functional blood vessels and/or regeneration of myocardium in damaged tissues.

Description

USE OF GEMIN A TO TREAT ISCHEMIC OR DAMAGED TISSUES

Field of the Invention This invention relates to methods of stimulating growth of functional blood vessels and/or regeneration of myocardium in damaged tissues, particularly damaged heart tissues and muscle tissues.

Background of the Invention Ischemic diseases, such as coronary heart disease and heart infarction remain the leading cause of death in the Western world, such as in America and in developed regions in Asia, such as in Hong Kong, and now becoming so in China as well. Ischemia is a condition in which blood flow (and thus oxygen) is restricted to a part of the body. Cardiac ischemia is the term for a lack of blood flow and oxygen to the heart muscle. Current available therapeutic approaches can only relieve symptoms and unfortunately, even with all the recent advances, cure of these kinds of ischemic diseases is difficult due to the impossibility to grow functional new vessels at an early stage in ischemic myocardium and to regenerate functional myocardium in myocardial infarction (Uchida et al, 1995; Lazarous et al., 1995; Pu, et al., 1993).

The conventional view was that cardiac myocytes do not divide in the adult for it was thought they are terminally differentiated cells comparable to neurons for their inability to regenerate and replace damaged tissue. Although evidences showed that a population of myocytes within the myocardium can and do replicate after infarction challenging the dogma that the heart is a postmitotic non-regenerating organ (Beltrami et al.m, 2001; Leferovich et al., 2001; Poss et al., 2002; Kajstura et al., 1998 & Schuster et al., 2004), the location of the newly regenerated cardiac myocytes occurs exclusively in the border zone adjacent to the infarct and in distant tissues where the blood supply is largely maintained. Left anterior descending coronary artery (LAD) ligation was used to induce myocardial infarction in the left ventricle and the myocardial infarction of this kind would induce complete damage to the myocardium affected and its interstitial tissue and blood vessels affected would be completely damaged and degraded. The only fate of myocardial infarction would be fibrous scar replacement significantly compromising the function of the affected heart.

The concept of therapeutic angiogenesis by augmenting the naturally occurring revascularizing process for the treatment of ischemic vascular diseases is a very attractive strategy. This may achieve more complete revascularization in patients with ischemic related diseases, such as coronary heart disease or heart infarction. For ischemic heart disease, apart from prevention, at present, blockages in the coronary arteries can only be relieved by surgery or angioplasty. There is no effective medicine that can stimulate the efficient growth of new blood vessels (angiogenesis) at early stage. Furthermore, after myocardial infarction, the myocardium is incapable of regenerating new cardiomyocytes to replace the lost myocardium. Scar tissues, which replace the necrosed myocardium, cause further deterioration in cardiac function. Therefore, an alternative revascularization strategy is required to treat ischaemia and stimulate replacement of damaged or lost heart muscle cells.

WO 03/04365 and WO 2004/052381 describe the use of an organic extract of Geum japonicum Thumb variant in stimulating growth of functional blood vessels and/or regeneration of myocardium or muscle fibers in damaged tissues, particularly damaged heart or muscle tissues. However, the exact compounds responsible for these functions are not known. It would not be possible to further study or enhance the above effects without knowing such compounds.

Objects of the Invention

Therefore, it is an object of this invention to identify the compounds responsible for the above effects in the organic extract of Geum japonicum Thumb variant. As a minimum, it is an object of this invention to provide the public with a useful choice.

Summary of the Invention

Accordingly, this invention provides [To be filled in after claims are consolidated]

Brief description of the drawings Preferred embodiments of the present invention will now be explained by way of example and with reference to the accompanying drawings in which:

Figure IA shows the extraction and isolation scheme of the compounds studied in this invention, and Figure IB shows the chemical structures of the active compounds Ga and Nif identified in this invention;

Figure 2 shows the growth of blood vessels in damaged tissues with the application of both Ga and Nif (GN), Ga, Nif, and the control (Con);

Figure 3 shows the results of does response experiments identifying the minimum amounts of the compounds required for producing significant proliferations of human coronary artery endothelial cells (HCAECs) and myobalst cells (C2C12);;

Figure 4 shows GN-mediated healing cascade of the infarcted myocardium at different time points;

Figure 5 shows the functional evaluation of GN treated heart infarction by echo- myocardiography; Figure 6 shows the development of cardiac differentiation of bone marrow mesenchymal stem cells (MSC) induced by GN;

Figure 7 shows the results of GN-mediated myocardial differentiation of MSC in SD rat heart infarction model; and

Figure 8 shows the results of GN-mediated healing cascade of severely injured muscles.

Detailed Description of the Preferred Embodiment

This invention is now described by way of example with reference to the figures in the following paragraphs.

Objects, features, and aspects of the present invention are disclosed in or are obvious from the following description. It is to be understood by one of ordinary skilled in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention, which broader aspects are embodied in the exemplary constructions. According to this invention, two compounds Ga and Nif were identified from the organic extract of Geum japonicum Thumb variant. The structures of these two compounds are shown in Figure 2.

General Use of Ga and Nif

Ga showed significant effect on early angiogenesis (functional blood vessel formation), and Nif displayed moderate angiogenetic effect and potent cardiomyogenic/myogenic effect in myocardial infarction or muscle injury animal models. When both of Ga and Nif (GN) were applied locally, it was found that the ischemic damaged myocardium underwent active neo-vascularization within 24-48 hours post-infarction with the formation of many functional blood vessels in the whole filed of the infarct. Newly regenerating cardiac myocytes were observed 3-4 days post infarction, forming many scattered Ki67 positively stained myocytes in the whole ares of the infarct including the central area and the border area of the infarct or within the surviving myocytes adjacent to the infarct border. Many new myocyte-like cells were also regenerated, clustered and formed myocardial-like tissue in the central area of the infarct between the newly formed blood vessels replacing almost half the total infarct volume after 7 days. In addition, many normally sized, Ki67 positively stained myocytes can be observed among the unaffected myocytes adjacent to the border region of the infarct. These newly regenerated myocytes might be derived from different sources compared with the regenerating myocardium in the central infarct with smaller sized myocytes.

The exact mechanisms by which GN promotes early reestablishment of blood supply network and functional myocardium regeneration in the whole area of infarct are not clear. GN may directly affect vascular endothelial and myogenic cells by promoting their proliferation and/or regeneration. It may also indirectly stimulate healing of infarcted heart by increasing the production of other growth factors, such as VEGF, or enhancing the action of growth factors delivered to the wound sites by platelets or macrophages. Furthermore, GN mediated active neo-vascularization in an early stage of the infarction may allow effective homing and differentiation of circulating stem cells, increasing the delivery rate of systemic growth factors and necessary components for effective healing to the infarcted area, enhancing removal of metabolic products or growth-inhibiting-factors, or inducing alteration in growth-factor receptors. According to the morphological features and the location of the newly regenerating myocardium-like tissue, the newly regenerated myocardium may come from two sources: (I) a sub-population of pre-existing cardiomyocytes, which are not terminally differentiated and possess the ability to divide when given the appropriate signal (Beltrami et aim, 2001; Leferovich et al., 2001; Poss et al., 2002 & Kajstura et al., 1998), or (II) from circulating stem cells, which may be attracted to the location of the infarct and induced to differentiate into cardiac myocytes or myocardium by GN. The demonstration of GN-induced cardiac differentiation of cultured mesenchymal stem cells supported the later explanation. The clusters of the newly regenerated myocardium-like tissues with smaller size observed inside the infarction area also supported the latter explanation. Furthermore, the early reestablishing blood supply network in infarct area is the prerequisite for homing circulating stem cells into infarct area and differentiating them into cardiac lineage. It is also possible that inhibition of fibrosis may also allow regeneration of myocyte.

Ga and Nif are not found to be only useful in treating cardiac diseases. Ga and Nif may be useful in treating damaged tissues requiring growth of blood vessels and/or regeneration of muscle cells, for example in treating damaged skeletal muscle tissues.

Use of Ga and Nif in Transplantation of Mesenchymal Stem Cells to Heart

GN may also be useful in induction of cardiac differentiation of mesenchymal stem cells in vitro and in the transplantation of mesenchymal stem cells for reconstituting functional myocardium and the growth of new coronary vessels in myocardial infarction. Transplantation of adult bone marrow-derived mesenchymal stem cells (MSCs) for treating myocardial infarction was shown to result in some angiogenesis and myogenesis. However, the location of the newly regenerated cardiac myocytes is restricted to the viable myocardium or occurs exclusively along the border zone adjacent to the undamaged myocardium where the blood supply is largely maintained (Poss et al., 2002; Lagasse et al., 2000 & Orilic et al., 2001). GN was found to be useful in: 1) enhancing survival capacity of the transplanted cells by up-regulated expression of endogenous Akt; 2) providing early improvement of microenvironment of the infarcted area induced by rapid revascularization, which would allow the trafficking of the transplanted cells to the whole infarcted area and support their survival locally, and 3) enhancing cardiomyogenic differentiation capacity of the transplanted cells by in vitro induction of cardiac differentiation of MSCs to form "cardioblasts" prior to transplantation. Since acute myocardial ischemia leads to rapid death of myocytes, the vascular structures and nonvascular components in the supplied region of the ventricle, regeneration of new cardiac myocytes replacing the infarcted myocardium in the central region through a sub-population of cardiac myocyte growth or transplantation of MSCs alone would seem to be impossible without early improvement of micro-environment locally. Therefore, the loss of myocardium, arterioles and capillaries in the central area of the infarct is irreversible resulting in scar formation eventually. Previous studies demonstrated that relatively few transplanted cells survive within the myocardium after injection (Toma et al., 2002). Thus far, regeneration strategies have not yet yielded replacement tissue with normal vascular/cardiomyocyte architecture. In Mangi et al., 2003, MSC modified with Akt delivered much promising results. However, the regenerating cardiac myocytes could only infiltrate from the border zone into the scarred area indicating that even transplanted MSCs with enhanced expression of exogenous Akt cannot survive in the absolute ischemic region. Evidences also showed that the MSC-derived regenerating cardiac myocytes scatter in the border zone adjacent to the undamaged tissue, but they are still difficult to cluster and form regenerating myocardium even in the border zones where the blood supply is largely maintained. This may suggest poor cardiomyogenic differentiation capacity of the survived transplanted MSCs. Three major requirements seem to be critical for an efficient trafficking, growth and trans-differentiation of transplanted MSCs into myocardium: 1) increased survival capacity of the donor cells; 2) early formation of new microvasculature to improve local micro-environment of the infarct that allows the efficient trafficking of transplanted cells, oxygenation and nutrient delivery; 3) the enhanced cardiomyogenic differentiation capacity of the transplanted cells.

To test this hypothesis, MSCs is mobilized in culture in the presence of Nif or butanol fraction of Geum Japonicum (BGJ) which contains both of Ga and Nif for 3 days to promote the over-expression of endogenous Akt. The cells were cultured for further 3 days in the presence of Nif or BGJ to induce their cardiac differentiation and form "cardioblast" with up- regulated endogenous Akt. The effects of early revascularization and cardiomyogenic differentiation of the transplanted "cardiobalsts" were assessed with rat myocardial infarction model. It was shown that after 2-3 days culture in the presence of the Nif or BGJ, the expression of endogenous Akt of the cultured MSCs was significantly up-regulated compared to their control cells in the absence of Nif or BGJ. Quantitative RT-PCR showed that 3 days culturing in the presence of Nif or BGJ resulted in significantly increased expression of the endogenous rat Aktl mRNA up to 3-4 folds compared to the non-treated control cells. Upon continuous culturing the Akt up-regulated MSCs for another 3-4 days, more than 80% of the cultured MSCs were positively stained with antibodies specific to early cardiac differentiating marker (MEF2), indicating their cardiomyogenic differentiation to form Akt up-regulated cardioblasts.

To validate the cardiac repair capacities of the Akt-cardioblasts in vivo, Akt- cardioblast transplantation in rat myocardial infarction model was studied. It was shown that numerous Dil-positively labeled cells on day 7 with the phenotype of cardiac myocytes were observed in the whole areas including central and border areas of the infarct. The positive DiI florescent signals from numerous regenerating cardiac myocytes distributed in the whole area of the infarct indicated their donor cardioblast origin. The Ki67 positive nuclei of these cardiac myocyte-like cells suggested that these transplanted cardioblast retain division capacity after transplantation. The micro-environment of the whole infarct area is improved, including the central and border areas of the infarct, by early growth of new blood vessels in affected areas that allows regeneration of cardiac myocyte clusters in central area of infarct since the transplanted cells in the central area of a myocardial infarct-the absolute ischemic region would die in hours if revascularization in the whole area of an infarct cannot be reestablished at early stage. In fact, many newly formed vessels were observed in the whole areas of the infarct 24 hours post infarction before any regenerating cardiac myocytes could be seen in the experiments of this invention. Many newly formed blood vessels and capillaries filled with blood cells were observed in the whole infarcted areas including the central areas and the border regions.

Astonishingly, numerous donor cell derived Ki67 and MHC positively stained cells were observed to organize into several cell clusters or myocardial-like tissue in the central area of the infarct in cardiobalst-transplanted hearts. Under high power microscope, typical morphology of myocardium were observed in these tissues, even though these regenerating myoblasts are smaller than undamaged cardiac myocytes and the newly regenerated cardiac myocytes along the border areas on the side of undamaged myocardium. The central regenerated and well organized myocardium may be the results of 1) up-regulated expression of endogenous Akt enhanced the survival of the donor cells in relative ischemic environment; 2) early revascularization enabled the efficient trafficking of the donor cells and 3) in vitro cardioblast trans-differentiation prior to injection promoted the cardiomyogenic differentiation. In contrast, MSCs-transplanted control hearts in which Nif is not applied, much less Ki67 positive cardiac myocytes were found along the border zones of a particular infarct, indicating that most of the donor cells died partially due to their weak survival capacity, and almost no central regenerating myocytes were observed probably due to both the weak survival ability of the donor cells and the failure of early reestablishing local micro- environment that blocks the migration of the donor cell to the central area of the infarct.

Further, DiI positive cells, some of which were Ki67 positively stained with the phenotype of myocardium, were found along the border on the infarction site forming highly organized cell clusters replacing the necrosed myocardium, indicating their high tendency towards cardiomyogenic differentiation compared with the limited number of scattered regenerating myocytes along the border zones in the control and in the previous reports using MSC transplantation approach (Poss et al, 2002; Lagasse et al., 2000 & Orilic et al., 2001). The blood supply network along the border zones was also found to be largely retained. If both cardiobalsts and MSCs have similar cardiomyogenic differentiation capacity in local micro-environment of myocardial infarct, similar regeneration capacity should have resulted in similar myocardial regeneration along border zones since the micro-environment and blood supply along the border zones should be about the same. The comparison between regenerating well organized myocyte clusters derived from cardiobalst transplantation and scattered regenerating cardiac myoctes derived from MSC transplantation in the region with similar micro-environment suggested that Nif-treated cardiobalsts possess superior cardiomyogenic differentiation capacity than that of non-treated MSCs.

In conclusion, this invention provides a new approach, namely applying Ga and/or Nif to the transplanted heart tissues, that can satisfy three critical requirements of 1) increased survival capacity of the donor cells; 2) early improvement of local micro-environment and 3) the enhanced cardiomyogenic differentiation capacity of the donor cells for repair of myocardial infarct, can result in good myocardial regeneration in the whole areas of an infarct. In comparison with the prior art: 1. transplanting cardiomyocytes or skeletal myoblasts (Yoo et al, 2000 & Taylor et al., 1998) failed to reconstitute myocardium and coronary vessels; 2. MSCs transplantation (Poss et al., 2002; Lagasse et al., 2000 & Orilic et al., 2001), only result in regeneration of scattered cardiac myocytes restricted along border zones; 3. transplanting MSCs with exogenous Aktl over-expression (Mangi et al., 2003) even though can increase survival capacity of the donor cells, therefore, resulting in much superior repair and regeneration of cardiac myocytes along border zones and infiltration of regenerating myocytes from border zone into the scarred area, this approach alone still could not regenerate well organized myocardium in the whole areas of the infarct including central area and border zones of the infarct.

The method of this invention satisfied the above three requirements, resulted in myocardial regeneration in whole areas of infarct and therefore, the superior repair of myocardial infarction.

The exact mechanisms by which the MSCs can be induced into cardiomyogenic differentiation by Nif to form cardiomyoblasts and the transplanted cardioblasts can promotes early reestablishment of blood supply network and functional myocardium regeneration in the whole area of infarct are not clear. The Nif or BGJ may trigger the expressions of some critical protein factor(s) in the cultured MSCs that induces a cascade of expressions of other protein factors driving the cardiomyogenic differentiation of cultured MSCs. This was indicated by the data of differential proteomic and micro-array studies, wherein about 16 (8 heparin-binding) differentially expressed protein spots or genes were identified during the process of trans-differentiation of MSCs into cardioblasts on daily basis. The transplanted cardioblast may not contribute to the cellular source of the newly formed vessels, but may directly affect endogenous vascular endothelial and myogenic cells by promoting their proliferation and/or differentiation or indirectly stimulate the process of early revascularization in infarcted myocardium by increasing the production of other growth factors through paracrine action. This may occur when the donor cardiolasts are forced to make their survival in relative ischemic environment as in the region of a myocardial infarct as demonstrated by Gnecchi et al., 2005.

Examples Example 1

During the course of screening for angiogenic reagents from Chinese herbal medicine, the methanol extract of Geum Japonicum thunb var (EGJ) has been identified that showed potent dual effects on stimulating early growth of new vessels both in ischemic heart muscles and infarcted heart muscles (< 48 hours), and on triggering myocardial regeneration in myocardial infarction.

The whole plant of Geum Japonicumδ Geum Japonicum collected from Guizhou Province of China in August was dried and percolated with Methanol at room temperature for 7 days. The extract is then dried under reduced pressure to yield a powder residue. The dried powder was suspended in H₂O and successively partitioned with hexane, ethylacetate and n- butanol respectively. All hexane, ethylacetate and n-butanol soluble fractions were filtered and evaporated under reduced pressure (50°C) yielding three different fractions, which were tested for their ability to stimulate angiogenesis and myogenesis in cell culture and in both muscle injury and myocardial infarction animal models. It was shown that n-butanol soluble fraction could enhance the proliferation of C2C12 myoblasts (ATCC) and HCAECs human coronary artery endothelial cells (Clonetics, Inc.) and promote early revascularization within 24 hours or 48 hours, and muscle fiber regeneration or myocardial regeneration in severe muscle injury or myocardial infarction animal models.

The active n-butanol soluble fraction was applied on a column of Sephadex LH-20 equilibrated with 10% methanol and eluted with increasing concentration of methanol in water, resolving 7 fractions. Further separation by liquid chromatography and NMR analysis demonstrated that there were two or three major compounds contained in each fraction of the sevens. One brown pure compound was isolated from fraction 3 displaying potent activity in enhancing angiogenesis and another white compound was isolated from fraction 6 showing significant effect on promoting myogenesis as well as some moderate angiogenic effect in cell culture systems and muscle injury/myocardial infarction animal models. The structural determinations of these two compounds by NMR analysis demonstrated that the compound, which showed potent angiogenic effect, isolated from fraction 3 is structurally corresponding to Gemin A (C₈₂Hs₆Os₂, Ga) and the other compound, which displayed potent cardiomyogenic/myogenic and moderate angiogenic effects, is niga-ichigoside Fl (C₃₆Hs₈O₁₁, Nif). The procedures of the isolation of the above two active compounds and the chemical structures of the two compounds are described as in Figures IA and IB.

As demonstrated in the process of bio-assay guided isolation of active compounds and shown in Figure 2, Ga mainly induced significant revascularization within 24 hours post muscle injury, but was less effective for myogenesis. Nif can also enhance significant myotube regeneration within 48 hours, but may be only moderate effective for early angiogenesis. Dose response experiment indicated that the minimum amounts of the methanol extract of the plant required for producing significant proliferations of HCAECs and C2C12 cells were approximately in the range of 20-80mg/ml (Figure 3: 3-1 and 3-2). n- butanol fraction of the methanol extract for eliciting similar effects as that of methanol extract was approximately 10-60 μg/ml; fraction 3 or 6 of the n-butanbl sub-fraction 5-30 μg/ml(Figure 3: 3-3); and 1-10 μg/ml of compound Ga or Nif were required for eliciting similar effect respectively (Figure 3: 3-4).

Repair of severe muscle injury or myocardial infarction requires both early angiogenesis and myogenesis. The combined use of Ga and Nif may create a suitable healing environment for severely damaged muscle and infarcted myocardium, hi this example, the compound Ga and Nif were combined in a 1:1 ratio (GN) and used to test their additive healing effects on myocardial infarction and muscle injury animal model (Figure 2, GN). The left anterior descending artery (LAD) of the experimental rats (300 g) was ligated. A single dose of GN (dissolved in 5% DMSO, 0.1 mg in 0.2ml) or control solution (5% DMSO, 02ml) was injected into the myocardium at the distal part of the ligated LAD respectively.

Evaluation of the dual effects of GN in myocardial infarct It was found that the infarct size in GN treated hearts (n=17) was on average approximately 1/3-1/2 times smaller than that in the control (n = 15) as measured by the volume of infarction in the left ventricular free wall associated with many newly formed vessels in the infarcted area on day 2 after ligation (Figures 4a & 4b). The capillary density in the infarct area of the GN treated myocardium was on average 9 per high power field (x 400) (HPF) derived from 6 randomly selected view fields of each slide and 17 slides in total from 17 GN treated hearts on day 2. hi contrast, fewer blood vessels (approximately 2 per HPF) with an inflammatory cell infiltration were observed in the controls on day 2.

On day 7 many newly formed blood vessels filled with blood cells remained in the infarction area including the central areas and the border regions of the infarct in GN treated animals (Figures 4c, 4d & 4e). More interestingly, numerous Ki67 positively stained cells with the phenotype of cardiac myocytes organized into several cell clusters or myocardial- like tissue in the central areas of the infarction in GN treated hearts were seen (Figure 4d). In the high power field, this myocardial-like tissue showed the typical morphology of myocardium, but these individual cells are smaller than surviving cardiac myocytes and the newly regenerated cardiac myocytes along the border areas. These newly formed myocardial- like tissues occupied about 49.3% of the total infarct volume on day 7 post-infarction. In contrast, in DMSO treated control hearts, there were only a few Ki67 positive cardiac myocytes (0-3) in each section of approximately 100 slices for a particular infarcted heart, and the infarction site was mainly occupied by fibrous tissue, Immunohistochemical staining with monoclonal antibodies against Ki67 exclusively and positively stains newly regenerated nuclei of cells and anti-MHC immunohistochemical staining only stains the cytoplasm of cardiac myocytes. Combining these double immunohistochemical staining procedures and morphological analysis the newly regenerated cardiac myocytes or myocardium could be identified. The nuclei of the undamaged myocardium surrounding the infarction areas were negatively stained for Ki67 but the cytoplasm was positively stained for MHC.

In the GN treated sample, numerous Ki67 positive cells with the morphology of myocardium were also found along the border on the infarction site forming highly organized cell clusters replacing the necrosed myocardium (Figures 4d & 4f). Approximately, 10-60 Ki67 positive cardiac myocytes could also be found along the proximal zone of surviving myocardium adjacent to the infarction border in each section (Figure 4f) of approximately 100 slices for a particular infarcted heart making on average approximately 5000 newly regenerated cardiac myocytes scattered among this region. The distal zone of surviving myocardium did not show any Ki67 positive cells. In the control heart no newly regenerated Ki67 and MHC positively stained cell clusters were found in the central and border areas of the infarcted regions. Instead, the infarction regions were mainly occupied by fibrous tissues (Figure 4g & 4h).

One month later, the morphological changes remained similar to those observations on day 7 post infarction. Many Ki67 and MHC positive myocytes with the phenotype of normal cardiac myocytes were found scattered in the surviving myocardium along the border zones of the infarction. Numerous Ki67 and MHC positive myocyte-like cells were highly organized into myocardial-like tissue that replaced the infarcted myocardium and therefore further reduced the infarct volume by 53-60% on average.

Functional evaluation of the GN treated hearts Histological changes accompanied by measurable functional improvement of the GN treated hears are shown in Figure 5. By 48 hours post-infarct, left ventricular ejection fraction (LVEF) in GN treated group had significantly improved (65.44 ± 2.41) by 21% compared to that (54.10 ± 2.27) in control hearts (p=0.001). Left ventricular fraction shortening (LVFS) in GN treated group was significantly higher than in control group (31.72 ± 1.67 vs. 24.41 ± 1.36, p = 0.002) and left ventricular end-diastolic dimension in GN treated hearts was smaller. These improvements were maintained to 1 -month post infarction.

Whatever the mechanisms by which GN exerts its potent and unique therapeutic effects, the above experiments prove that cardiac myocytes can divide, the infarction can be repaired by the newly regenerated myocardium and the function of the infarcted heart can be significantly improved in the early stages after infarction and onwards, making myocardial regeneration a real possibility. Thus, GN appears to have highly beneficial effects in promoting both early reestablishment of the impaired blood supply network and regeneration of functional myocardium replacing the necrosed heart tissue in myocardial infarction.

Example 2

SD rat bone marrow-derived MSCs were isolated and cultured for 14 days. The MSCs were cultured in the presence of GN (10 μg/ml) for 3 days respectively, and the expression of Akt was assessed by immunocytochemistry and quantitative RT-PCR. The cells with up- regulated Akt expression were cultured for further 3 days in the presence of GN. Their cardiac differentiation was assessed by immunocytochemistry and Western blot against heart type early marker MEF2. The MSC-derived cardiac differentiating cells with over-expression of Akt were termed Akt-cardioblasts. The MSCs, which were cultured for 6 days in the absence of GN, were used as control. Both Akt-cardioblasts and control MSCs were labeled with CM-DiI in culture (Silva et al., 2005). It was shown that after 2-3 days culture in the presence of the GN, the expression of endogenous Akt of the cultured MSCs was significantly up-regulated (Figure 6 Akt) compared to their control cells in the absence of GN. Quantitative RT-PCR showed that 3 days culturing in the presence of GN resulted in significantly increase of the endogenous rat Aktl mRNA up to 3-4 folds compared to the non-GN-treated cells. The enhanced cardiomyogenic differentiation of these Akt-upregulated MSCs was indicated by the positive cardiac markers (heart type myosin heavy chain, MEF2 and troponin I (Figure 6 Tro I & MHC). The cardiac repair capacities of the Akt-cardioblasts in vivo was validatedin myocardial infarction animal model. The Sprague-Dawley (SD) rats were used and all animal procedures were approved by the University Animal Committee on Animal Welfare. Myocardial infarction was induced by permanent ligation of LAD coronary artery. The DiI labeled Akt-cardioblasts (5 X 10⁵) suspended in saline were injected into three sites of the distal myocardium (the ischemic region) of the ligated artery immediate after the ligation (test group). The control rats were injected with an equivalent amount of DiI labeled control MSCs suspended in saline at the same location and timing. Vascular density was determined on day 7 post-infarction from histology sections by counting the number of vessels within the infarct area and expressed as the number of vessels per high power field (HPF). The infarct size was quantified by triphenyl-tetrazolium chloride (TTC) staining. The regeneration of myocardium was assessed on day 14 post-infarction from histology sections by evaluation of the volume of the regenerating myocardium in the central area of the infarct. The sections were immunohistochemically stained with both rat-specific Ki67 and myosin heavy chain (MHC) antibodies sequentially to double confirm the regenerating myocardium. The homing, survival, proliferation and cardiomyogenic differentiation of the transplanted cardioblasts were identified by the positive signals of either Dil-florescence and by antibody immunostaining for Ki67 and MEF2 in sections made from the hearts on day 7 and 14 post infarction respectively.

It was shown that numerous Dil-positive cells on day 7 (Figure 7A) with the phenotype of cardiac myocytes were observed in the whole areas including central and border areas of the infarct. The positive green florescent or DiI florescent signals from numerous regenerating cardiac myocytes distributed in the whole area of the infarct indicated their donor cardioblast origin. The Ki67 positive nuclei of these cardiac myocyte-like cells suggested that these transplanted cardioblast remain the division capacity after transplantation (Figure 7D). The whole area, including central and border areas of the infarct, and location of the regenerating cardiac myocyte clusters is believed to be due to the micro-environmental improvement by early revascularization, since the transplanted cells in the central area of a myocardial infarct-the absolute ischemic region would die in hours. In fact, many newly formed vessels were observed in the whole areas of the infarct 24 hours post infarction before any regenerating cardiac myocytes could be seen. Many newly formed blood vessels and capillaries filled with blood cells were observed in the whole infarcted areas including the central areas and the border regions (Figures 7 A, 7B, 7D). The density of the newly formed vessels in infarcted area of the cardioblast transplanted myocardium was on average 7-9 per high power field (x 400) (HPF) on day 7, however, no Dil-positive vessels were observed indicating that the cellular source of the newly formed vessels may not be contributed by the donor cells, but the donor cardioblasts may trigger and stimulate the process of early revascularization via a paracrine action, hi comparison, only approximately 2-4 vessels per HPF were observed in the MSC transplantated controls on day 7 (Figure 7B). More interestingly, numerous donor cell derived Ki67 and MHC positively stained cells organized into several cell clusters or myocardial-like tissue in the central area of the infarct in cardiobalst transplanted hearts were seen (Figure 7C & 7D).

hi the high power field, this myocardial-like tissue showed the typical morphology of myocardium, but these regenerating myoblasts are smaller than undamaged cardiac myocytes and the newly regenerated cardiac myocytes along the border areas on the side of undamaged myocardium. These newly regenerated myocyte-like cells were highly organized into myocardial-like tissue, which occupied averagely 48% of the total infarct volume on day 7 and replaced the infarcted myocardium by 68% cm average on day 14 post-infarction in the test group.

The central regenerated and well organized myocardium shows that 1) up-regulated expression of endogenous Akt enhanced the survival of the donor cells in relative ischemic environment; 2) early revascularization enabled the efficient trafficking of the donor cells and 3) in vitro cardioblast trans-differentiation prior to injection promoted the cardiomyo genie differentiation. In contrast, in MSCs transplanted control hearts there were much less Ki67 positive cardiac myocytes along the border zones in each section of approximately 100 slices for a particular infarct indicating that most of the donor cells died partially due to their weak survival capacity, and almost no central regenerating myocytes were observed probably due to both the weak survival ability of the donor cells and the failure of early reestablishing local micro-environment that blocks the way of the donor cell trafficking to the central area of the infarct. Numerous DiI positive cells, some of which were Ki67 positively stained, with the phenotype of myocardium were also found along the border on the infarction site forming highly organized cell clusters replacing the necrosed myocardium (Figure 7B & 7D). This indicates high tendency towards cardiomyogenic differentiation compared with the limited number of scattered regenerating myocytes along the border zones in the control using MSC transplantation approach, although the blood supply network along the border zones largely retained. If both cardiobalsts and MSCs have similar cardiomyogenic differentiation capacity in local micro-environment of myocardial infarct, similar regeneration capacity should have resulted in similar myocardial regeneration along border zones. It is because the micro- environment and blood supply along the border zones should be about the same. MHC and Akt immunohistochemical staining demonstrated that many of the regenerating cardiac myocytes in the whole areas of the infarct remain positively stained by the antibodies specific to MHC (Figure 7E) and Aktl (Figure 7F). The nucleus location of the positive staining suggests that the up-regulated Aktl is in active form. The comparison between regenerating well organized myocyte clusters derived from cardiobalst transplantation and scattered regenerating cardiac myoctes derived from MSC transplantation in the region with similar micro-environment suggested that cardiobalsts possess superior cardiomyogenic differentiation capacity than that of non-treated MSCs.

In conclusion, the application of GN to transplanted heart tissues can satisfy three requirements: 1) increased survival capacity of the donor MSCs; 2) early improvement of local micro-environment by early revascularization and 3) the enhanced cardiomyogenic differentiation capacity of the donor cells for repair of myocardial infarct, and result in superior myocardial regeneration in the whole areas of an infarct. This results in myocardial regeneration in whole areas of infarct and therefore, the significant repair of myocardial infarction.

Example 3

Severe muscle injury healing is a complicated and dynamic process involving complex mechanisms that can be accelerated by growth factors. It is believed that an appropriate combination of growth factors may promote regenerative potential of severely damaged muscles resulting in improved healing and that the interaction of growth factors and certain tissue-repair cells such as satellite cells, fibroblasts, and endothelial cells plays a key role in the healing process (Menetrey et al., 2000; Yamada, 2000 & Chan et al., 2005). Muscle injury caused by trauma, crush injury, excessive exercise, or disuse is common, particularly in professional sports players. Muscle has a limited potential regenerating ability, however, muscle healing following severe injury is slow and incomplete in most situations, resulting in fibrous tissue replacement compromising the function of the muscle affected. At present, there is no effective therapeutic measure that can be taken to promote significant skeletal muscle regeneration after muscle severe injury (Nikolaou et al., 1987; Almekinders

6 Gilbert, 1986 & Reddy et al., 1993).

Wound healing requires the presence of multiple factors, the factors required for neovascularization, cellular proliferation and differentiation. In the case of muscle healing including skeletal muscles, smooth muscles and heart muscles, the necessary prerequisite for effective healing requires the combined actions of early reestablishing blood supply networks and myogenesis. Therapeutic neo-vascularization at wound sites, where the previous blood vessels have been damaged, can improve and enhance the delivery of oxygen for cell respiration and metabolism, requisite growth factors and other necessary components for supporting cellular proliferation and tissue regeneration in the wound sites and the removal of metabolite wastes and growth-inhibiting factors away from the wound sites. Therefore, the healing process of damaged muscle tissue can be summarized into three phases: 1) the initial destruction phase with concomitant haematoma formation, myofiber necrosis, and inflammation; 2) the repair phase, consisting of phagocytosis of the necrotized tissue and regeneration of the myofibers by activating, proliferating and differentiating the quiescent satellite cells; 3) the remodeling phase, where the regenerated myofibers mature and the function of the repaired muscle is restored (Jarvinen et al., 2000 & Wright-Carpenter et al., 2004). Based on the features of GN provided in the first two examples, in the present example, the potent effects of GN on early angiogenesis and myogenesis were tested with a severe injured muscle animal model.

Muscle injury animal model and treatment protocol

Seventy male 200-250-g Sprague-Dawley (SD) rats were used. The rats were subjected to strain-induced muscle injury created by a transection with surgical scissors on the tibialis anterior. After closing and proper suturing of the wound GN in 5% DMSO (0.1ml, containing 0.3mg GN) was injected into the damaged area of the injury immediately (test group). Another 70 rats were subject to the same procedure of muscle injury but injected with an equivalent amount of 5% DMSO at the same location and timing as control group. Ten rats from the test group and 10 rats from the control group were sacrificed on day 1, 2, 3, 4, 5,

7 and day 14 respectively after operation. The tibialis anterior muscles containing the injured areas of the sacrificed rats at different time points were removed and washed with PBS respectively. AU the specimens harvested were fixed in neutralized 10% formaldehyde for histological study. Estimation of GN-induced time-dependent healing effects

The muscle sections from both test and control groups on day 1, 2, 3, 4, 5, 7 and 14 post-injury were used for evaluation of the dual effects of GN on healing of the severely damaged muscles. To assess the effect of GN on early neovascularization in the wound area, vascular density was determined on day 2 post-injury from histology sections by counting the number of vessels within the injury area using a light microscope under high power field (HPF) (x 400). Muscle regeneration capacity was assessed by muscle histological sections obtained on day 7 post injury from both groups, which were stained with hematoxylin and eosin. Slides were analyzed by determination of the number of the newly regenerating centronucleated (CN) myotubes versus per 1000 μm wide injury gap. The size of the healthy non-damaged muscle fibers was found to vary between 80-120 μm and that of the regenerating CN cells between 20-70 μm. The numbers of regenerating myofibers within 1000 μm wide injury gap for each muscle sample were averaged. The potential of muscle regeneration was expressed as the percentage of the number of normal muscle fibers, which are accommodated by 1000 μm wide region (number of regenerating myotubes per 1000 μm wide injury gap /10 X 100%).

As observed in Figure 8: Id (one day after cut injury to the tibialis anterior and a subsequent treatment with a single injection of GN in 5% DMSO), many newly formed blood vessels were formed, but no newly regenerated myotubes could be observed in the wound field. It appears that although revascularization could actively start within 24 hours injuryD myotube regeneration requires more time. On day 2 post injury and GN treatment, some scattered newly regenerated long thin myotubes along with many newly formed vessels were observed in the wound fields (Figure 8:2d). More and more regenerating and elongated myotubes could be seen on day 3 post injury (Figure 8:3d). More regenerating and elongated myotubes aligned in a correct direction with the existing undamaged muscle fibers forming long thin myotube bundles replacing the damaged myofibers or damaged part of the muscle fibers on day 4 post injury (Figure 8:4d). The regenerating myotubes grew thicker than day 4 and some of the newly formed myotubes were found to fuse with the partially damaged muscle fibers precisely to repair the damage on day 5 post injury (Figure 8:5d). The regenerating myotubes continued growing and clustering to form more matured myotube bundles replacing most of the damaged myo fibre bundles in the wound sites, and between the regenerating myotube bundles many matured blood vessels could be found to support the regeneration (Figure 8:6d). On day 7, the regenerating myotubes further elongated and well- aligned with centrally located and PCNA positive nuclei, and some proliferated PCNA positive satellite cells were also detected along the more matured regenerating myotubes. The gap of the injured muscle fibers was bridged in a correct orientation (Figure 8:7d). There were on average 11-13 regenerating long thin myotubes observed in per 1000 mm injury gap (Figure 8:4d to 7d). On day 14, the elongated, well aligned and relatively matured newly regenerating myofibers completely bridged the gaps formed by the muscle injuries (Figure 8:14d). The contour of the cutting lines could still be recognized by the differentially stained muscle fibers between pre-existing muscle fibers and newly regenerating muscle fibers probably due to the content difference of the proteins between the two different staged muscle fibers.

In the control experiment, the wound field was infiltrated with many inflammatory cells and no newly regenerating myotubes were found with almost no or much less new blood vessels formed in 5% DMSO control animals on day 3 post injury. On day 7 post injury, the wound fields of the control muscles were full of inflammatory cells and fibrous tissue replacement, and no satellite cells could be observed. On 14 days post injury the wound fields of control were healed with fibrous scar; the faster growing fibrous tissues replaced the damaged muscle fibers and the wound gaps were contracted (Fig. 8: 14d-Con).

hi this example, GN showed the potent dual and additive effects of angiogenesis and myogenesis on muscle injury repair. The GN-mediated healing cascade of severely damaged muscles could be deciphered from the time course of muscle healing with our current animal model experiments. When a single injection of GN was applied subcutaneously over the tibialis anterior right after the damage to the muscle, it was found that the severely damaged muscles underwent active neo-vascularization within 24 hours post-injury forming many functional blood vessels in the wound field. Newly regenerating myotubes could be observed 48 hours post injury forming scattered long thin myotubes along the newly formed blood vessels. More regenerating long thin myotubes were produced, clustered and formed myotube bundles in correct orientation replacing the damaged muscle fiber bundles or fusing with the partially damaged myofibers to repair them on day 5-7; the regenerating myotubes further grew, matured and completely bridged up the gaps of muscle injuries on day 14. In contrast, there were almost no newly regenerated functional vessels and myotubes could be observed but inflammatory cells infiltration in the control on day 3, inflammatory cell infiltration and fibrous tissue replacement on day 7 and fibrotic scar formation on day 14. This example proves that the additive effects of early angiogenesis and myogenesis can induce complete healing of severe muscle injury, which was previously impossible.

While the preferred embodiment of the present invention has been described in detail by the examples, it is apparent that modifications and adaptations of the present invention will occur to those skilled in the art. Furthermore, the embodiments of the present invention shall not be interpreted to be restricted by the examples or figures only. It is to be expressly understood, however, that such modifications and adaptations are within the scope of the present invention, as set forth in the following claims. For instance, features illustrated or described as part of one embodiment can be used on another embodiment to yield a still further embodiment. Thus, it is intended that the present invention cover such modifications and variations as come within the scope of the claims and their equivalents.

Claims

CLAIMS:

1. The use of a compound having the formula:

in stimulating growth of at least one of functional blood vessels and myocardium in ischemic or damaged tissues of a subject.

2. The use of Claim 1 , wherein the subject is a human being.

3. The use of Claim 1, wherein the compound is in a concentration of 0.1 tolO,000 μg/ml.

4. The use of Claim 1, wherein the ischemic tissues are ischemic heart tissue, ischemic limbs, ischemic muscles or ischemic bones.

5. The use of Claim 1, wherein the damaged tissues are damaged heart tissue, damaged muscle tissue or damaged bone tissue.

6. A method for stimulating growth of at least one of functional blood vessels and myocardium in ischemic or damaged tissues of a subject, including the step of applying a compound having the following structure to the ischemic or damaged tissues:

7. The method of Claim 6, wherein the subject is a human being.

8. The method of Claim 6, wherein the compound is in a concentration of 0.1 to 10,000 μg/ml.

9. The method of Claim 6, wherein the ischemic tissues are ischemic heart tissue, ischemic limbs, ischemic muscles or ischemic bones.

10. The method of Claim 6, wherein the damaged tissues are damaged heart tissue, damaged muscle tissue or damaged bone tissue.

11. The use of a compound having the following formula:

in the manufacturing of a medicament for stimulating growth of at least one of functional blood vessels and myocardium in ischemic or damaged tissues of a subject.

12. The method of Claim 11 , wherein the subject is a human being.

13. The use of Claim 11, wherein the compound is in a concentration of 0.1 to 10,000 μg/ml.

14. The use of Claim 11, wherein the ischemic tissues are ischemic heart tissue, ischemic limbs, ischemic muscles or ischemic bones.

15. The use of Claim 11, wherein the damaged tissues are damaged heart tissue, damaged muscle tissue or damaged bone tissue.