CN114606307B - Application of fibroblast activation protein-alpha as drug target - Google Patents
Application of fibroblast activation protein-alpha as drug target Download PDFInfo
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- CN114606307B CN114606307B CN202011519396.2A CN202011519396A CN114606307B CN 114606307 B CN114606307 B CN 114606307B CN 202011519396 A CN202011519396 A CN 202011519396A CN 114606307 B CN114606307 B CN 114606307B
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention provides application of fibroblast activation protein-alpha serving as a target spot in screening medicaments for treating myocardial infarction or ischemia reperfusion injury. The drug is a drug that inhibits the expression level of fibroblast activation protein-alpha. The drug is a drug that inhibits the activity of fibroblast activation protein-alpha. The invention provides application of fibroblast activation protein-alpha gene expression and protein activity as drug targets in screening drugs for treating myocardial infarction or ischemia reperfusion injury. According to the invention, through systematic analysis of the expression condition of Fap, the expression of Fap is firstly interfered from the gene level, the Fap can be found to be a potential target point for myocardial infarction and ischemia reperfusion injury treatment, and then a series of pharmacological interventions are carried out, so that the protease activity inhibiting Fap has a good treatment effect in myocardial infarction and ischemia reperfusion injury treatment.
Description
Technical Field
The invention belongs to the field of biological medicine, and relates to application of a medicine target spot in screening and treating medicines for myocardial infarction, in particular to application of fibroblast activation protein-alpha serving as a target spot in screening medicines for treating myocardial infarction or ischemia reperfusion injury.
Background
Cardiovascular diseases are the leading lethal factor worldwide at present, and according to the summary of Chinese cardiovascular disease report 2019, the mortality rate of acute myocardial infarction in China is rapidly increased from 2005. The treatment measures aiming at myocardial infarction comprise medicament intervention modes such as blood circulation reconstruction, antiplatelet, blood fat control and the like, but the current comprehensive treatment scheme does not lead the death rate of the cardiovascular disease of China to be reduced, and along with the aggravation of aging population, the morbidity and the death rate of myocardial infarction still continue to be increased year by year.
Myocardial infarction is one of the most serious and most fatal diseases among cardiovascular diseases, and a large number of mature myocardial cells with contractile capacity die rapidly after myocardial infarction, resulting in serious damage to the structure and function of the heart. Since the capacity of myocardial cell proliferation is weak and the damaged heart cannot be repaired rapidly, fibroblasts play a great role in the repair of injury after myocardial infarction. The fibroblast cells residing in the heart are rapidly activated after being stimulated by local necrosis factors, metabolites, inflammatory factors and the like after myocardial infarction and begin to proliferate in large quantity, and the activated and proliferated heart fibroblast cells can secrete a large quantity of extracellular matrix proteins, so that granuloma is formed at the damaged part, angiogenesis is promoted, and finally, a fibrous scar is formed, thereby repairing the damaged heart and protecting the structure and function of the heart. Fibrosis and angiogenesis play an important role in the repair of myocardial infarction. Excessive fibrosis may lead to diastolic dysfunction, reduced ventricular compliance, leading to long-term heart failure; however, insufficient fibrosis may also lead to heart failure and scar enlargement. Therefore, it is important to effectively control the function of fibroblasts after myocardial infarction.
Fibroblast activation protein-alpha (Fibroblast activation protein alpha, fap) is a type two transmembrane integral glycoprotein located on chromosome two of human, mouse, bovine, sheep, human and mouse sharing 89% amino acid sequence. Fap exists mainly on fibroblast membrane, and consists of 6, 18 and 736 amino acids in cytoplasmic, transmembrane and extracellular structures respectively, and has collagenase and dipeptidase activities, degradable dipeptide, type I collagen and the like. Previous researches show that Fap is mainly and highly expressed in tumor-associated fibroblasts (Cancer Associated Fibroblasts, CAFs), and is widely involved in the growth, invasion, metastasis and tumor extracellular matrix reconstruction, angiogenesis and immune escape processes of tumors and in the formation and regulation of tumor microenvironment. Fap expression is also closely related to injury repair and the like, and plays an important role in rheumatoid arthritis, osteoarthritis, pulmonary fibrosis, liver fibrosis and other related diseases. In 2015, tillmanns et al found that Fap could be used as a marker for activating fibroblasts after myocardial infarction for the first time; in 2017, bauersachs et al detected the change of Fap concentration in peripheral blood of patients suffering from acute ST elevation myocardial infarction (STEMI) for the first time, and proposed that the change of Fap is related to myocardial injury and inflammatory response for the first time, and can be used as biomarker for diagnosis of myocardial infarction. In 2019, aghajanian et al sequenced heart tissues of 238 normal adults and heart failure patients, found that Fap was the fibroblast marker protein with the highest expression level in heart failure patients such as ischemic heart failure, hypertrophic cardiomyopathy, dilated cardiomyopathy and the like, and Monika Litvi ň ukov et al mapped the most complete human heart cell map so far in 2020, and the results showed that Fap specifically marked a group of heart fibroblasts. In summary, in the previous researches, fap is mostly taken as a surface marker protein of cardiac fibroblasts, the action mechanism of Fap protein in cardiovascular diseases is not clear, after the Fap positive cardiac fibroblasts are directionally killed by using a CAR-T method, myocardial fibrosis can be obviously relieved, the systolic and diastolic functions of the heart are improved, and the Fap marked fibroblasts are suggested to have important pathophysiological effects, but at the same time, the CAR-T treatment method also causes serious adverse reactions such as anemia, weight loss, muscle and bone loss and the like, so that how to effectively regulate the functions of the Fap positive fibroblasts is important.
After myocardial infarction, promoting angiogenesis is an important therapeutic means for promoting myocardial repair after myocardial infarction, preventing excessive reconstruction of heart and correcting heart failure, and Fap is taken as a protease, has the activities of dipeptidase and endopeptidase, and the action mechanism of Fap probably plays a role due to enzyme cleavage of a substrate. Then, whether Fap has an important role after myocardial infarction, whether its role results from the proteolytic cleavage or breakdown function of Fap, resulting in an enhancement or attenuation of the biological protective effect of the protective polypeptide (or inhibitory polypeptide) is not known. Previous studies have shown that Fap cleavage substrates include classical collagen type I, gelatin, and the like, and in addition, fap can cleave human α2-antiplastmin, neuropeptide Y, P, polypeptide YY, and Brain Natriuretic Peptide (BNP) in vitro studies. Wherein, BNP has strong regulation and control effect on pathological states after heart stress, and BNP can further influence cGKI, akt, erk/2 and other channels by activating cGMP, so that angiogenesis is influenced, and the enzyme digestion of human BNP by Fap can greatly lighten the biological activity of the BNP. However, it is currently unclear whether Fap can cleave BNP effectively in an in vivo state, whether BNP has similar biological activity after cleavage, and whether Fap inhibition in vivo has therapeutic potential. The discovery of the basic research provides a new thought for researching the functions of the Fap protein and provides an important theoretical basis for the follow-up effective regulation and control of the functions of the Fap protein.
Disclosure of Invention
Aiming at the technical problems in the prior art, the invention provides application of fibroblast activation protein-alpha serving as a target point in screening medicaments for treating myocardial infarction or ischemia reperfusion injury, and the application of the fibroblast activation protein-alpha serving as the target point in screening medicaments for treating myocardial infarction or ischemia reperfusion injury aims to solve the technical problems of poor effect and limited means for treating myocardial infarction or ischemia reperfusion injury in the prior art.
The invention provides application of fibroblast activation protein-alpha serving as a target spot in screening medicaments for treating myocardial infarction or ischemia reperfusion injury.
Further, the drug is a drug that inhibits the gene and protein expression levels of fibroblast activation protein- α.
Further, the drug is a drug that inhibits the protease activity of fibroblast activation protein- α.
The invention provides application of fibroblast activation protein-alpha gene expression as a drug target in screening drugs for treating myocardial infarction or ischemia reperfusion injury.
The invention provides application of protease activity of fibroblast activation protein-alpha as a drug target in screening drugs for treating myocardial infarction or ischemia reperfusion injury.
The invention provides application of an inhibitor of targeted fibroblast activation protein-alpha in preparing a medicament for treating myocardial infarction or ischemia reperfusion injury.
The invention provides application of targeted fibroblast activation protein-alpha in preparing a biomarker for diagnosing and prognosis judging myocardial infarction patients.
In view of the fact that the current comprehensive treatment means of cardiovascular diseases still cannot effectively reduce the mortality, the invention discovers a novel treatment target point, which is used for improving the treatment means of serious cardiovascular diseases such as myocardial infarction, ischemia reperfusion injury and the like, improving the effective rate and the success rate of treatment and reducing the mortality of the cardiovascular diseases.
The application method of the targeted inhibition fibroblast activation protein-alpha (Fap) in myocardial infarction treatment mainly comprises the following steps:
fap is highly expressed in situ in adult pathologic heart reconstruction, the concentration of free Fap in serum is reduced, and the concentration of free Fap in serum has good correlation with indexes such as heart function, heart injury and the like, so that Fap can be used as a biomarker for diagnosing and prognosis judging pathologic heart reconstruction, especially myocardial infarction patients.
The invention intervenes the functions of Fap protein through various methods, particularly including knockout and knockout on the gene level and inhibition on the pharmacological level, and researches show that the expression and the functions of Fap can be intervened to effectively play a good role in treating myocardial infarction and ischemia reperfusion injury.
Fap inhibition protects heart function after stress mainly because its enzymatic activity is inhibited, resulting in protective polypeptides that are not clearly functional. For example, BNP is a novel endogenous substrate of Fap, which is found by the invention, and a plurality of substrates such as type I collagen, alpha 2-antiplastmin, neuropeptide Y, P substances and the like which are found in the past can possibly change expression after heart stress injury, and the intervention of Fap functions can influence the activity of the substrates to play a potential protective role, so that Fap is an important intervention target found in the invention.
Compared with the prior art, the invention has obvious technical progress. The invention provides a brand-new intervention target for treatment after myocardial infarction and ischemia reperfusion injury, wherein the intervention target is fibroblast activation protein alpha (Fibroblast activation protein alpha, fap), and the intervention mode is mainly to knock down the expression and inhibit the protein function. Through a series of in vivo and in vitro experiments, a new endogenous substrate BNP of Fap is found, and the potential therapeutic effect of the Fap inhibitor is further clarified through comparison with the marketed positive medicines, so that the Fap inhibitor has stronger conversion potential to a certain extent, and can effectively protect heart reconstruction and functional damage after myocardial infarction and ischemia reperfusion injury and promote angiogenesis in an infarcted area.
Drawings
FIG. 1 shows the expression of Fap in adults and mice and its correlation with prognosis in example 1.
FIG. 2 shows the effect of Fap gene knockout on mouse growth and cardiac function under steady state conditions in example 2.
FIG. 3 shows the protective effect of Fap gene knockout on cardiac function and cardiac structure after myocardial infarction in example 2.
FIG. 4 shows the therapeutic effect of small molecule compounds of example 3 on inhibition of Fap.
FIG. 5 shows a Fap inhibitor-specific assay in example 4.
FIG. 6 shows the therapeutic effect of Fap intervention on ischemia reperfusion injury in example 5.
FIG. 7 shows the effect of Fap intervention on type I collagen and angiogenesis in example 6.
FIG. 8 shows in vitro verification of the cleavage of Fap and BNP in example 7.
FIG. 9 shows the effect of BNP on the tube formation of HUVECs in example 8.
FIG. 10 shows the effect of Fap on BNP in example 8 to promote the pipelining and migration of HUVECs.
FIG. 11 shows the therapeutic effect of FAPi on substrate BNP knockout mice in example 9 in comparison with the positive drug LCZ 696.
Detailed Description
Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.
Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention; in the description and claims of the invention, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed in the present invention employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, and related arts. These techniques are well described in the prior art.
Fibroblast activation protein abbreviation Fap
FAPi abbreviation for fibroblast activation protein inhibitor
Naolinatriuretic peptide abbreviation BNP
Human umbilical vein endothelial cell abbreviation HUVECs
Acute myocardial infarction abbreviated AMI
IHF for short of ischemic heart failure
Acronym HCM for hypertrophic cardiomyopathy
Ejection fraction abbreviation EF
Short axis shrinkage abbreviation FS
Left ventricular end diastole inner diameter abbreviation LVID; D
Left ventricular end diastole volume abbreviation LVEDV
End diastole ventricular septum thickness abbreviation IVS, D
The end-diastole left post-ventricular wall thickness is abbreviated as LVPW.
EXAMPLE 1 confirmation of Fap high expression in adult and mouse pathologically reconstituted hearts
First, we collected heart tissues of healthy donors, ischemic heart disease patients and hypertrophic heart disease patients, and analyzed the mRNA levels and protein levels of Fap in heart tissues, and the results showed that the expression levels of mRNA and protein in heart tissues of ischemic heart failure (Ischemic heart failure, IHF) and hypertrophic heart disease (Hypertrophic cardiomyopathy, HCM) patients were significantly increased as compared with healthy donors (FIG.1A-1C), staining of adult myocardial tissue using Masson and Fap immunofluorescence revealed a significant increase in the degree of cardiac fibrosis in IHF and HCM patients, and a significant increase in Fap expression (fig. 1D-1G). Subsequently, by examining serum of normal subjects, AMI patients and IHF patients, the content of free Fap in serum was measured, and it was found that the content of free Fap in serum of AMI patients and IHF patients was decreased compared with that of normal subjects, and at the same time, the activity of serum Fap enzyme was also significantly decreased in AMI and IHF patients (FIG. 1H-1I). After linear regression analysis of the concentration of free Fap in serum and the ejection fraction (Ejection Fraction, EF) of the cardiac contractility index of the patient, we found that the concentration of free Fap in serum was positively correlated with the EF values of AMI and IHF patients (fig. 1J-1K). Subsequently, we divided AMI patients into high concentration Fap group and low concentration Fap group by median serum free Fap concentration, and D-SPECT detected all patients, and patients in low concentration Fap group were found to have higher ischemia scores (SRS score, SSS score) under resting and loading conditions, and greater overall reperfusion defect (Total perfusion defect, tpd%) (fig. 1L-fig. 1O). To further study the function of Fap, we also observed the expression of Fap after myocardial infarction in mice, and the results showed that, after myocardial infarction in mice, the infarct areaFapThe mRNA expression level gradually increased in the early stage to peak 7 days after myocardial infarction, followed byFapmRNA expression levels gradually decreased to almost normal levels by 28 days (FIG. 1P). Since previous studies showed that Fap is a surface marker protein of activated cardiac fibroblasts, we found that by co-staining with the previously recognized surface marker protein α -SMA of activated cardiac fibroblasts, fap and α -SMA were significantly highly expressed in cardiac tissue 7 days after mouse myocardial infarction, and there was much co-localization of both (fig. 1Q), suggesting that Fap expression levels were elevated after myocardial infarction, and a population of specific activated cardiac fibroblasts was labeled.
Example 2 UsingFapFunctional verification of Fap in knockout mice
To further explore the specific function of Fap after myocardial infarction, we purchased with Jackson labFapGene knockout mouseFap -/- ) And performing subsequent function verification. First, we analyzeFapThe effect of gene knockout on the growth and development of mice under normal conditions, and found that, in the followingFapAfter the gene was knocked out, the mice had no obvious differences in weight, organ weight and tibia length (FIGS. 2A-2F), suggesting Fap -/- Does not affect the growth and development of mice. Subsequently, we for 8 weeksFap -/- The mice were stained with Masson's heart section, and found to be associated withFapThere was no significant difference in the degree of myocardial interstitial fibrosis between the two mice expressing normally (FIGS. 2G-2H). At the same time, we are toFap -/- As a result of WGA staining of heart sections of mice and control mice, no significant difference in the size of cardiomyocytes was observed in the steady-state condition (FIGS. 2I-2J). In terms of cardiac structure and function, we found by cardiac hypermeasurement that both had similar diastolic ventricular septum thickness (IVS; D), diastolic left post-ventricular wall thickness (LVPW; D), cardiac Ejection Fraction (EF), short axis systolic (FS), and end-diastolic left ventricular volume (LVEDV) (FIGS. 2K-2O). Thus, we determined that at steady state, knockout of the Fap gene had no significant effect on mouse growth, extent of myocardial interstitial fibrosis, cardiomyocyte size, cardiac structure and function.
To further investigate whether Fap has biological function under stress, we utilizedFap -/- Myocardial infarction model of mice and foreleg permanent ligation, forFapThe function is explored, and the result shows that the myocardial infarction of the miceFapThe gene knockout can effectively slow down ventricular remodeling and cardiac hypofunction after myocardial infarction of mice, and cardiac ultrasonic results show that,Fap -/- mice had better left ventricular contractile function 1, 3, 7, 14 days after myocardial infarction, with EF and FS both significantly higher than the control group (fig. 3A-3C), while,Fap -/- mice also had lower left ventricular end-diastole volumes, smaller heart/body weight ratios, 7 days post myocardial infarction,Fap -/- mouse IVS; D, LVPW; D was higher, all of which indicateFapGene knockoutThe heart of the mice is less reconstructed after the removal,Fapknock-out is effective in preserving the structure and function of the heart (fig. 3D-3f,3 k). In order to better observe the scar and fibrosis condition after the myocardial infarction of the mice, the heart tissues 7 days after the myocardial infarction of two groups of mice are subjected to section staining, and the result shows thatFap -/- The mice had smaller scar area and higher degree of interstitial fibrosis (FIGS. 3G-3J). In order to evaluate the size of scar tissue as comprehensively as possible after myocardial infarction in mice, we obtained two groups of hearts 7 days after myocardial infarction, fixed with 4% PFA, and then soaked in 25% lugol's solution for 24 hours for Micro-CT scan, and the results showed that, from the whole point of view,Fap -/- mice also have smaller overall scar volumes (fig. 3L-3M), and thus we believe that Fap gene knockout plays a protective role after myocardial infarction, leading to better pathological remodeling of the heart, better scar repair in the acute phase, and thus better systole function in mice after myocardial infarction.
EXAMPLE 3 pharmacological inhibition of Fap enzyme Activity can effectively protect cardiac Structure and function in mice after myocardial infarction
To further investigate whether Fap exerts its heart regulating function in dependence on enzyme activity, we used Fap enzyme activity specific small molecule inhibitor (FAPi) Ac-Gly-Boropro (an acetylated glycine borated proline, purchased from MCE (MedChemExpress) company, cat# 886992-99-0) to intervene on Fap enzyme activity to verify if Fap enzyme activity inhibitor has a protective effect similar to Fap gene knockout.
First, we tested the dose curve of FAPi, and based on the enzyme activity Ki values of Ac-Gly-Boropro, we selected 0.15 mg/kg, 0.5 mg/kg and 1.5 mg/kg as 3 treatment dose groups, the fixed treatment time was 7 days, mice after myocardial infarction were given daily intraperitoneal injection, and the mice were analyzed for mortality, cardiac structure and function, histology, organ weight, etc. (specific experimental procedure as shown in FIG. 4A), and as a result, we found that the 0.5 mg/kg FAPi treatment dose group was effective in reducing mice mortality after myocardial infarction (FIG. 4B), and we found that the EF value was higher for the 0.5 mg/kg treatment group by measuring the heart contraction function (FIG. 4C). Subsequently we cut and stained the mouse hearts 7 days after the FAPi treatment, and found that there was no significant difference in scar area between the vehicle treatment group and the 0.5 mg/kg FAPi treatment group near the ligation site level, but the FAPi treatment group had more interstitial fibrosis, and the ratio of heart/body weight was also significantly reduced after the FAPi treatment compared to the control group (fig. 4D-4G), suggesting that heart reconstruction was less after the FAPi treatment, and that the 0.5 mg/kg FAPi treatment gave the optimal treatment effect.
Subsequently, we further tested the time profile of the FAPi treatment, and in the case of defining the optimal treatment dose, we determined the treatment dose to be 0.5 mg/kg, the treatment time was set at 7 days, 14 days and 28 days, and the heart structure and function data, histological data, organ weight data, etc. of the mice were collected (specific experimental procedure is shown in fig. 4H), and as a result, it was found that the heart contractile function could be effectively protected after 7 days of the FAPi treatment, the proportion of heart reconstruction of the mice was greatly reduced with the treatment time, and the left ventricular diastolic volume of the mice was significantly reduced from that of vehicle after 14 days and 28 days of treatment (fig. 4J). Subsequently we further analyzed the degree of mouse cardiac interstitial fibrosis at various time points of the FAPi treatment, which indicated that the degree of mouse cardiac interstitial fibrosis gradually increased with the extension of the treatment time (fig. 4K-4L).
Example 4 FAPi acts in a Fap-dependent manner and specifically inhibits Fap enzymatic Activity
To further exclude that the Fap inhibitor Ac-Gly-Boropro exerts cardioprotective effect after myocardial infarction in a manner independent of Fap enzyme activity, we further selected a small molecule compound SP-13786 (purchased from MCE (MedChemExpress) company, cat# 1448440-52-5) with a completely different mechanism of action from Ac-Gly-Boropro as a novel Fap enzyme activity inhibitor and treated the mouse myocardial infarction model, and found that SP-13786 was effective in protecting the heart contractile function of mice after 7 days of treatment, SP-13786 treated mice EF, FS were higher (FIGS. 5A-5C), inhibiting pathological reconstitution of the heart of mice after myocardial infarction, as evidenced by a smaller end-diastole volume of the left chamber of the SP-13786 treated mice (FIG. 5D). The brand new inhibitor SP-13786 can also play an effective role in protecting after myocardial infarction, which suggests that the inhibition of the activity of the Fap enzyme has potential therapeutic value.
To further exclude that small molecule inhibitors may nonspecifically target inhibition of other enzyme activities when inhibiting Fap enzyme activity, we therefore utilized small molecule compounds to further inhibit the enzyme activity of other enzymesFap -/- The specificity of the small molecule compound inhibitor is detected in mice, and after 7 days of FAPi treatment, we find that the FAPi treatment group of wild mice after myocardial infarction can effectively protect the heart contraction function of the mice and improve the pathological reconstruction of the heart, but the FAPi treatment is performed after 7 days of FAPi treatmentFap -/- The absence of additive effects in mice (FIGS. 5E-5H) suggests that FAPi-specific enzyme activity on Fap plays a therapeutic role without off-target effects of inhibitors.
Example 5FapGene knockout and inhibition of Fap enzyme activity also exert protective effects in ischemia reperfusion injury
Ischemia reperfusion injury due to rapid revascularization following myocardial infarction is more common in clinical practice. Unlike permanent ligation-induced persistent ischemia, more oxidative stress and other injuries may be caused by blood reperfusion in the ischemia reperfusion process, so as to further simulate the actual conditions encountered in clinical treatment, we further explore whether Fap gene knockout and Fap enzyme activity inhibition can also play a protective role in a mouse myocardial ischemia reperfusion injury model. Firstly, we explore the influence of Fap gene knockout and FAPi treatment on ischemia dangerous areas and infarct areas after ischemia reperfusion injury for 72 hours by Evans blue/TTC staining method, and found that the Fap gene knockout and FAPi treatment can reduce the ischemia reperfusion injury area of mice, compared with a control group,Fap -/- group and FAPi treated group mice had smaller ratios of ischemic risk/left ventricular area, infarct size/ischemic risk area (fig. 6A-6D). And (3) withAt the same time, we also observed by cardiac ultrasoundFap -/- Group and FAPi treated group mice have altered cardiac function and cardiac structure, which indicatesFap -/- Mice in the group and the FAPi treated group had better contractile function at the time points of 7 days and 28 days, with significantly higher EF and FS values, less ventricular remodeling and less left end diastole volume in both groups (FIGS. 6E-6H). Subsequently we performed microtome Masson staining of heart tissue in three groups of mice, which showed that after 7 days of ischemia reperfusion injury,Fap -/- both the group and the FAPi treated group showed more myocardial interstitial fibrosis, suggesting stronger cardiac scar repair (fig. 6I-6J). Finally, we have shown that by analyzing the ratio of heart weight to body weight of mice, the results suggest that after 7 days and 28 days of ischemia reperfusion injury,Fap -/- both the group and the FAPi treated group had smaller heart weight/body weight ratios (fig. 6K).
Example 6 Fap Gene knockout and inhibition of Fap enzyme Activity promote type I collagen deposition and angiogenesis
In the acute phase of myocardial infarction, deposition and angiogenesis of ECM such as collagen have important roles in promoting cardiac function recovery and protecting cardiac structure and function, and since Fap can effectively degrade type I collagen, we further explore whether Fap gene knockout and Fap enzyme activity inhibition can influence type I collagen deposition and angiogenesis at the edge of myocardial infarction of mice after myocardial infarction. After immunofluorescent staining of heart sections of wild-type, fap knockout mice and FAPi-treated mice, we found that Fap knockout mice and FAPi-treated mice had significantly increased deposition of type I collagen and angiogenesis in the border region of myocardial infarction, suggesting that Fap affected type I collagen and angiogenesis following myocardial infarction (fig. 7A-7D).
EXAMPLE 7 determination that Fap can be effective for cleavage of mouse BNP
BNP plays an important role in regulating and controlling various pathological and physiological stresses in the heart, and previous researches show that Fap can carry out effective enzyme digestion on human BNP, so that the biological activity of BNP is reduced, and the BNP loses the cardiac regulation and control effect after stress. But the mouse BNP isIt is not clear whether the cleavage by Fap can be effectively performed until now, and in order to further explore the cleavage effect of Fap on mouse BNP, we first go through the cleavage of wild type,FapImmunofluorescent staining of heart sections of knockout mice and FAPi-treated mice, foundFapThe expression of BNP in myocardial infarction marginal area in heart sections of gene knockout mice and FAPi treated mice is obviously increased, which suggests that BNP is possibly a substrate of Fap endogenous, then, we further verify the expression quantity of BNP in heart tissues in marginal area after myocardial infarction of three groups of mice through western blot, and the result shows thatFapBNP expression levels were significantly increased in heart tissue of knockout mice and FAPi-treated mice (FIGS. 8A-8D). Thus, to further verify that Fap was co-incubated with both proteins and polypeptides in vitro, we first fixed the dose of Fap used, found that in the 10 ul cleavage system, the 10 ng recombinant Fap protein was effective to cleave mouse BNP after 48 hours, and that 48 hours co-incubation by means of a gel blue dye resulted in a faint strip of mouse BNP (fig. 8E-8F), and then we tested the cleavage of mouse BNP at different doses within 24 hours, and as a result found that with increasing Fap dose, cleavage of mouse BNP gradually increased, and that Fap continued to co-incubate with mouse BNP for 24 hours after pretreatment of Fap protein by the Fap inhibitor, and that Fap was effective to cleave mouse BNP was lost (fig. 8G-8H).
Example 8 in vitro angiogenesis promoting effects of mouse BNP can be blocked by Fap
To further explore the effect of Fap-digested BNP on angiogenesis, we studied the effect of mouse BNP on the ability of HUVECs to tube using human umbilical vein endothelial cells (Human umbilical vein endothelial cells, HUVECs). First, we tested the effect of different concentrations of mouse BNP on the ability of HUVECs to promote tube formation, we set four dose groups of veccle, 1 nM,10 nM,100 nM, and after 16 hours, we analyzed the effect of mouse BNP on HUVECs to promote tube formation, with both 1 nM and 10 nM dose groups significantly promoting HUVECs to be able to promote tube formation, with 1 nM and 10 nM dose groups having more chambers formed by HUVECs to form a larger total chamber area and a longer total branch length, with 10 nM dose groups having better effects than veccle 1 (FIGS. 9A-9B). Subsequently, we further tested the effect of 10 nM mouse BNP on HUVECs at different time points in the tube formation, and as a result found that mouse BNP significantly promoted tube formation by HUVECs at 8 hours, 16 hours and 24 hours compared to vehicle, with more connection points, more lacuna formation, larger total lacuna area, and longer total branch length in the group treated with mouse BNP, with the two groups differing most significantly at 16 hours (fig. 9C-9G).
After determining the optimal dose and optimal treatment time, we selected 10 nM mouse BNP for 16 hours on HUVECs, observed the effect of vehicle, recombinant Fap protein, mouse BNP, co-incubation of mouse BNP with Fap on HUVECs tube formation, and found that Fap had no significant effect on HUVECs tube formation compared to vehicle, mouse BNP was effective in promoting HUVECs tube formation, and this tube formation promoting effect disappeared after co-incubation of Fap with mice, which co-incubation had no effect on HUVECs tube formation (fig. 10A-10B).
In addition to the effect of the vascular formation on the tube formation capacity of HUVECs, migration capacity also has an important effect on vascular formation, and we have therefore further explored the effect of mouse BNP on tube formation of HUVECs. Through transwell's experimental system we found that recombinant Fap protein and 10 nM mouse BNP were effective in promoting migration of HUVECs, but their ability to promote migration of HUVECs disappeared after Fap co-incubation with mouse BNP (fig. 10C-10D). To further clarify the effect of Fap and mouse BNP on HUVECs migration ability, we further validated the results of transwell using scratch experiments, consistent with the results of transwell that Fap and mouse BNP alone both promote healing of scratch, but the ability to promote both disappeared after co-incubation (fig. 10E-10F), further validated the regulation of BNP to promote HUVECs migration ability by Fap, suggesting that mouse BNP is an in vitro substrate for Fap.
Example 9 determination of failure of FAPi treatment in BNP knockout mice
To further verify that BNP is an endogenous substrate for Fap, we clarified the therapeutic effect of FAPi in BNP knockout mice by modeling myocardial infarction on substrate-BNP knockout mice and administering Fap enzyme activity inhibitor treatment (specific experimental procedure as shown in fig. 11A), and the study results showed that FAPi can significantly improve cardiac structure and contractile function in wild-type mice, and that FAPi-treated mice alone had higher EF, FS, smaller left end-diastolic inner diameter and left end-diastolic volume, but no significant therapeutic effect after 7 days of FAPi treatment in BNP knockout mice (fig. 11B-11F). Subsequently, we compared the therapeutic effect parallel to that of the FAPi by using the marketed and enzyme-cleavable BNP enkephalinase inhibitor LCZ696 (fig. 11G) as a positive drug, and the results show that both the FAPi and the LCZ696 can protect the heart function and the heart structure after myocardial infarction of mice, and the combined treatment of the FAPi and the LCZ696 has no superposition effect (fig. 11H-11L), further suggesting that the two act on a common target point, namely BNP, and providing more powerful evidence for that the BNP is an endogenous substrate of the Fap.
Claims (2)
1. Use of fibroblast activation protein-alpha as a target in the selection of a medicament for the treatment of myocardial infarction or post-ischemic reperfusion injury, the medicament being a medicament that inhibits the expression level of fibroblast activation protein-alpha or a medicament that inhibits the activity of fibroblast activation protein-alpha.
2. Use of an inhibitor of targeted fibroblast activation protein-alpha in the preparation of a medicament for the treatment of myocardial infarction or after ischemia reperfusion injury, said inhibitor being inhibiting the expression level of or inhibiting the activity of fibroblast activation protein-alpha.
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| Changes in concentrations of circulating fibroblast activation protein alpha are associated with myocardial damage in patients with acute ST-elevation MI;Jochen Tillmanns等;Int J Cardiol;第232卷;第155-159页 * |
| Fibroblast activation protein alpha expression identifies activated fibroblasts after myocardial infarction;Jochen Tillmanns等;Journal of Molecular and Cellular Cardiology;第第87卷卷;摘要，材料与方法，结果 * |
| 急性心肌缺血猝死心肌中FAPα和TGF-β1的表达;袁翔天等;法医学杂志;第28卷(第1期);第18-20页 * |
| 成纤维细胞激活蛋白在小鼠心脏组织中时程表达变化及内肽酶活性测定;廉虹等;中国分子心脏病学杂志(第03期);第60-63页 * |
| 王晓良.应用分子药理学.北京：中国协和医科大学出版社,2015,第222-223页. * |
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