Garcinoic Acid Improves Cardiac Function and Enhances Angiogenesis Following Myocardial Infarction

Cardiac ischemia impairs angiogenesis in response to hypoxia, resulting in ventricular remodeling. Garcinoic acid (GA), the extraction from the plant garcinia kola, is validated to attenuate inammatory response. However, the role of GA in heart failure (HF) and neovascularization after myocardial infarction (MI) is incompletely understood. The present study is striving to explore the role of GA and the potential mechanism of which in cardiac function after MI. SD rats were randomized into sham group, MI+vehicle group, and MI+GA group in vivo. Human umbilical endothelial cells (HUVECs) were cultured in vehicle or GA, and then additionally exposed to 2% hypoxia environment in vitro. MI rats displayed a dramatically reduced myocardial injury, cardiac function and vessel density in the peri-infarcted areas. GA delivery markedly improved cardiac performance and promoted angiogenesis. In addition, GA signicantly enhanced tube formation in HUVECs under hypoxia condition. Furthermore, the expressions of proangiogenic factors HIF-1α, VEGF-A and bFGF, and pro-angiogenic proteins phospho-VEGFR2Tyr1175 and VEGFR2, as well as phosphorylation levels of Akt and eNOS were increased by GA treatment. In conclusion, GA preserved cardiac function after MI probably via promoting neovascularization. And the potential mechanism may be partially through upregulating the expressions of HIF-1α, VEGF-A, bFGF, phospho-VEGFR2Tyr1175 and VEGFR2 and activating the phosphorylations of Akt and eNOS.


Introduction
Acute myocardial infarction (MI) is one of the chief causes of global morbidity and mortality (Cao et al., 2014). Occlusion of coronary arteries, which leads to insu cient oxygen and nutrient supply, is the major pathology of MI and it is by which the ischemia progress into necrosis. Restoration of myocardial blood supply in necrotic or peri-infarct areas as soon as possible is bene cial to myocardial repair after MI (Shah & Mann, 2011). Early reperfusion of the acute occlusive coronary artery greatly improves the prognosis of patients with MI, thereby reducing myocardial necrosis (Yellon & Hausenloy, 2007). This strategy is regarded as the most effective method for the treatment of acute MI in clinical studies. However, it needs more justi cation whether such treatments towards microvascular rarefaction and cardiac dysfunction in the ischemic heart can contribute to an effective myocardial ischemia-reperfusion.
Proangiogenic therapy has theoretically appeared a promising strategy for anti-ventricular remodeling after MI, but current clinical trials reported little positive news (Tongers et al., 2011). Notwithstanding we face such a dilemma in vascular regeneration therapy, the challenge must be overcome before therapeutic angiogenesis becomes a pragmatic therapeutic methodology.
In the early stage of in ammation, macrophages and neutrophils clear necrotic cardiomyocytes and promote the proliferation of endothelial cells (ECs) and broblasts to form vascularized granulation tissue, but then it can cause apoptosis of these cells to form collagen-rich scars, resulting in microvascular rarefaction (Frangogiannis, 2006). Inhibition of in ammatory responses can signi cantly enhance healing after MI by increasing neovascularization in peri-infarcteded areas (Tang et  . Garcinia kola can also attenuate doxorubicin-induced cardiotoxicity and protect against ischaemia/reperfusion injury (Oyagbemi et al., 2017;Oyagbemi et al., 2018). In addition, recent researches have demonstrated that puri ed garcinoic acid (GA) from Garcinia kola nut seeds, as a major bioactive component in the seed, has an antiin ammatory effect and can inhibit the occurrence of atherosclerosis (Wallert et al., 2019). However, up to now, it is unknown whether administration of GA has an cardioprotective effect on cardiac dysfunction and neovascularization after myocardial ischemia.
In the present study, we investigated the role of GA in myocardial angiogenesis of peri-infarcteded areas and cardiac function,,as well as examined the potential mechanisms through both vivo and vitro methods.

2.1.Experimental animals and treatments
All adult male Sprague-Dawley (SD) rats (weighing 180-200 g) were acquired from the Animal Center of the Renmin Hospital of Wuhan University. All animal care and experimental procedures were approved by the Guidelines for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (revised 2011) and in compliance with the Animal Care and Use Committee of Renmin Hospital of Wuhan University. All rats were maintained under speci c pathogen-free (humidity 50 ± 5%; temperature 20-22 °C) with a 12-hour light/dark cycle.
Rats were subjected to either a ligation of left anterior descending branch to make MI model or a sham operation according with previous method (Yuan et al., 2016a). The rats were randomly divided into three groups: a sham operation group (sham), a MI group (MI+vehicle) and a garcinoic acid treatment (GA)+MI group (GA+MI). We administered GA (1 mg/kg; Sigma-Aldrich, St. Louis, MO, USA) or vehicle (PBS+DMSO) by intraperitoneal (i.p.) injection weekly for 2 weeks (Wallert et al., 2019). All the specimens were collected from the peri-infarcted areas (< 2 mm outside the infarct).

Cell culture and treatments
Human umbilical endothelial cells (HUVECs; Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were used for all in vitro experiments. HUVECs were cultured in Dulbecco's modi ed Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS,GIBCO,USA) / 1% penicillinstreptomycin (100 U/mL) in a cell incubator with humidi ed 95% O 2 and 5% CO 2 atmosphere at 37°C for normoxia condition. Hypoxia condition was performed with hypoxic gas mixture (2% O 2 , 5% CO 2 and 93% N 2 ) from cell incubator. Before hypoxia, HUVECs were pretreated in different concentrations of GA (1μM, 2.5μM and 5μM) or vehicle for 24 hours.

2.4.Cell viability
The cell viability was evaluated by the Cell Counting kit (CCK-8) assay (Dojindo, GB707, Japan). The cells were treated with various concentrations of GA and vascular endothelial growth factor (VEGF) and/or hypoxia and incubated with 10 μL CCK8 solution on a 96-well microplate reader for 2 hours,.The absorbance was detected at 450 nm wavelength by an ELISA reader.

2.5.Tube formation assays
After allowing the BD Matrigel TM Basement Membrane Matrix to settle for 30 min in a 37℃, 5% CO2 incubator, HUVECs from different groups were seeded on the layer of polymerized gel on a 96-well plate, hypoxia+VEGF was used as a positive control. 12 hours after incubation, the formation of a capillary-like structure was recorded using microscope and images taken by light microscopy (ECLIPSE 80i; Nikon, Japan). Tube formation was quanti ed by using image-analysis software (Image J, version 1.40g, NIH).

2.6.Enzyme linked-immunosorbent assay (ELISA)
Plasma creatinine kinase-MB (CK-MB; Jiancheng Bioengineering Institute, Nanjing, China) and cardiac troponin I (cTnI; Life Diagnostic, Pennsylvania, USA) from jugular vein blood at various time points (0, 7 and 14day) were determined with ELISA detection kits as per manufacturer's instructions. After treatment for 2 weeks, the serum was collected to detect the concentration of BNP with a BNP ELISA detection kit (BD Biosciences, San Jose, CA) according to the manufacturer's instructions.

2.7.Masson's Trichrome
Maximum section of heart was xed in 4% formaldehyde for 24h, after which it was embedded in para n, and cut into 5 µm thick slices. The slices were stained with Masson trichrome staining for evaluation of infarcted size in the heart. The brosis areas were observed using a light microscope (Olympus D72, Japan) .The infarcted size was calculated as the percentage of the positively stained area of the total myocardial tissue, and then analyzed by using Image J software.

2.8.Immuno uorescence analysis
The dissected hearts in the peri-infarcteded areas were xed in 4% phosphate-buffered formalin, and then the samples were embedded in para n to cut into 5 μm sections. To evaluate vessel density and proliferation, alpha smooth muscle actin (α-SMA)/CD31 and ki67/CD31 co-immunostaining was performed. After blocking with 10% goat serum, the sections were incubated in α-SMA, ki67 and CD31 primary antibody (Abcam, Cambridge, UK) overnight and subsequently incubated with Alexa-Fluorcoupled secondary antibodies, then counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen, Carlsbad, CA, USA). The images were obtained by using a uorescence microscope (OLYMPUS, Tokyo, Japan) and then analyzed by using Image J software.

2.10.Statistical analysis
The data analyses were performed with the GraphPad Prism 6.0. All data were expressed as mean ± SEM. Comparisons for multiple-group were analyzed using ANOVA with the Tukey's test. A value of P< 0.05 was deemed statistically signi cant.

3.1.GA treatment reduces myocardial injury and promotes recovery of cardiac function after MI
To determine the effects of GA on infarcted size, Masson-trichrome staining was performed, the result showed that the infarct size was not obviously reduced with GA treatment in post-MI rats (Fig. 1A, B).
Furthermore, the levels of CK-MB and cTnI was considerably higher in the MI group at the 7 day post-MI, whereas GA signi cantly decreased this effect, but no signi cant difference was observed between the MI and GA+MI groups at the 14 day post-MI (Fig. 1C, D), suggesting a reduction in cardiac injury post-MI to some extent. To clarify whether GA therapy could improve cardiac function in MI rats, echocardiographic examination was carried out at day 14 after intervention. Compared with the sham groups, MI groups markedly induced cardiac dysfunction, as indicated by the distensible LVEDD and decreased EF and FS, which were partially restored to normal cardiac contractility and LV geometry in GA+MI supplement groups (Fig. 1E-H). Furthermore, cardiac dysfunction was also assessed by the serum levels of BNP by Elisa analysis, and we observed that GA could attenuate the MI-induced upregulation of BNP levels (Fig.  1I). Therefore, we concluded that GA treatment alleviated MI-induced myocardial necrosis and cardiac dysfunction in rats.

3.2.GA therapy boosts post-MI angiogenesis
Cardiac vessel density in the border zone was quanti ed with percentage of positive areas 14 days after operation through immunohistochemistry double-staining with α-SMA (a smooth muscle marker) and CD31 (an endothelial cell marker), Ki67 (a cell proliferation marker) and CD31. As expected, evidenced by the number of positive cells of CD31 (CD31 + ), as well as α-SMA + / CD31 + cells, MI rats showed a signi cant vessel density rarefaction in peri-infarcteded areas compared with sham groups whereas administration of GA increased the density of the mature blood vessels post-MI ( Fig. 2A, C, D). Cell nuclear antigens Ki67and CD31 were co-immuostained to determine endothelial proliferation. The number of Ki67 + cells and Ki67 + / CD31 + was signi cantly higher in the group receiving GA than that in the group receiving vehicle (Fig. 2B, E, F). These data indicated that neovascularization in the border zone of hearts at day 14 after MI was enhanced by GA treatment.

3.3.GA upregulates the expression of pro-angiogenic proteins and p-Akt, p-eNOS in post-MI hearts
To elucidate the underlying mechanisms, we examined the effects of GA on the expression levels of several pro-angiogenic proteins including HIF-1α, VEGF-A and bFGF, and related signaling molecules p-Akt and p-eNOS. In vivo study, compared with the sham groups, MI groups had no effect on the production of HIF-1α, VEGF-A and bFGF in peri-infarcted areas of hearts. Treatment with GA induced a signi cant increase of these proteins compared with the vehicle-treated MI groups (Figs. 3A-D). Despite a slight increase of p-VEGFR2 (Tyr1175), it had no signi cant downregulation in the peri-infarcted myocardium of MI rats compared to sham rats. Treatment with GA markedly enhanced VEGFR2 phosphorylation. Furthermore, GA could increase the expression of VEGFR2 in infarcted hearts (Figs. 3E-G). The proangiogenic protein p-Akt was signi cantly upregulated in MI rats compared with the sham groups, while p-eNOS had no difference in the two groups. However, GA treatment stimulated the expressions of both p-Akt and p-eNOS (Figs 3H-J). Thus, vivo study showed that GA enhanced the phosphorylation of Akt and eNOS through upregulation of HIF-1α, VEGF-A and bFGF, the effect of which possibly promoted post-MI neovascularization.

3.4.GA enhances the tube formation ability of HUVECs subjected to hypoxia
Initially, to con rm the possible cytotoxicity of GA towards HUVECs, we assessed cell viability using a CCK8 assay in exist of GA and/or hypoxia. We found that pretreatment with different concentration of GA for 30 h has no signi cant effect on HUVECs viability under normoxia condition. As expected, VEGF which served as a positive control boosted cell proliferation, (Fig. 4A). As shown in Fig. 4B, hypoxia for 6 h can induce the decline of cell activities. An obvious increase in cell proliferation was observed in GAtreated (2.5μM and above) cells in the presence of hypoxia. In vitro, HUVECs tubule formation ability was signi cantly depressed when HUVECs were subjected to hypoxia in Matrigel culture, whereas GA (2.5μM and above) could notably promote the formation of a tubular capillary-like network (Fig. 4C, D). The results illustrated that GA made for the formation of vascular tube-like structure under ischemia and hypoxia condition.

3.5.GA activates the expressions of angiogenesis proteins in vitro possibly via enhancing the Akt/eNOS signaling pathway
We further illuminated the mechanisms underlying pro-angiogenic phenomenon in vitro assay. Consistent with above methods, we pretreated HUVECs with different concentrations of GA for 24h, and then cultured them in hypoxia or normoxia for 6 h. The data showed that hypoxia induced higher HIF-1α and VEGF-A expressions in hypoxia HUVECs than in vehicle, while the expression of bFGF presented no signi cant difference between hypoxia and vehicle groups. As expected, the production of HIF-1α, VEGF-A and bFGF was further dramatically augmented by different concentrations of GA treatment which had different e cacy, though (Fig. 5A-D). Exposure to GA rapidly resulted in a striking increase in phosphorylation of VEGFR2 within only 10 min. With the prolongation of time, the degree of p-VEGFR2 was attenuated (Fig. 5E, F). Pre-treatment with the GA (2.5 μM) pronouncedly increased the expression of VEGFR2 in hypoxia conditions (Figs. 5G, H). In addition, phosphorylation of Akt was notably activated in the hypoxia groups compared with the vehicle groups. Besides, p-eNOS did not signi cantly appear in hypoxia-treated HUVECs. The supply of GA further enhanced the degree of Akt and eNOS phosphorylation (Fig. 5I-K). Consistent with the vivo alterations, we found that the upregulation of HIF-1α, VEGF-A and bFGF affected by GA treatment was associated with activating p-Akt and p-eNOS in vitro study.

Discussion
In the present study, we observed that GA alleviated myocardial injury and cardiac dysfunction and promoted vascular regeneration in the infarcted border zone following MI. Supplementation of GA raised the endothelial cells viability and boosted tube formation in hypoxia-treated HUVECs. Furthermore, we also provided evidences that HIF-1α, VEGF-A, bFGF, and p-Akt/p-eNOS signaling pathways played an essential role in the angiogenesis, Therefore, our current ndings approved that GA might be an effective therapeutic agent against cardiac injury.
In ammatory response, compensatory hypertrophy of muscle and imbalance of vascular regression in the myocardium play an important role in cardiac injury and repair (Shiojima et al., 2005;Liehn et al., 2011). Promoting in ammatory cells release anti-in ammatory cytokines and angiogenic factors, which are critical to vanish the in ammation and promote angiogenesis (Nahrendorf et al., 2007;Vannella & Wynn, 2017). It is well established that neovascularization in the peri-infarcted areas after MI is conducive to preserve cardiac function, thus reducing adverse left ventricular remodeling (Frangogiannis, 2012). Actually, except for delivering oxygen and nutrients, the crosstalk of endothelial cell/cardiomyocyte also regulates the cardiac contractility, giving rise to the protection of cardiomyocytes after ischemia in an NO-independent manner (Winegrad et al., 1998;Leucker et al., 2011). As expected, GA, an extraction from Garcinia kola nut, enhanced vascular density and angiogenesis in peri-infarcted areas of hearts post-MI. Moreover, considering that ECs proliferation is indispensable in the early stage of sprouting angiogenesis, we underwent cell viability assay in vitro.. And the result is in line with the proangiogenic effect of GA treatment in vivo. The protective effect of GA on cardiac function after MI is probably due to its already well-de ned anti-in ammatory effect and its pro-angiogenic effect found in the present study.
Regulating speci c transcriptional responses of mammalian cells to hypoxia is overwhelmingly mediated by HIF-1 which is composed of an HIF-1α and an HIF-1β subunit (Wang et al., 1995). HIF-1α, as an upstream initiator of hypoxia and ischemia-induced angiogenesis, mediates cellular adaptation to hypoxia and regulates angiogenesis and therein has been demonstrated to be a pivotal regulator for gene expression, including those encoding angiogenesis cytokines such as VEGF and bFGF. (Pugh & Ratcliffe, 2003;Calvani et al., 2006). Meanwhile some studies have demonstrated that VEGF treatment promotes neovascularization, increases vessel density, and ameliorates post-MI myocardial repair (Giacca & Zacchigna, 2012;Cheng et al., 2013). The presence of HIF-1α-bFGF pathway enhances survival and sprouting of ECs under hypoxic conditions. Spatiotemporal delivery of bFGF promotes cardiac tissue vascularization and improves cardiac function following MI (Calvani et al., 2006;Fan et al., 2019). Intriguingly, our data con rmed that GA can promotes the expression of HIF-1α, VEGF and bFGF in vivo and in vitro experiments. Previous studies have demonstrated that VEGFR-2 played a vital role in angiogenesis and activation of VEGFR-2 promoted ECs growth, migration and tube formation. VEGF-A, as a crucial pro-angiogenic factor through VEGFR2 signal pathway can awake a variety of biological activities, including endothelial cells maturation, angiogenesis and arteriogenesis in response to hypoxia or healing. (Ferrara et al., 2003;Jin et al., 2018). Upregulation of these pro-angiogenic proteins was probably the main reason why GA can promote endothelial cell morphogenesis in vitro and angiogenesis in vivo.
Akt kinases regulate cellular survival, proliferation, metabolism and have been a rmed to play a vital role in revascularization (Chen et al., 2005). Consistent with previous reports, our data showed that the degree of phosphorylation of Akt increased in 2 weeks after MI (Rengo et al., 2012). While further upregulated p-Akt was one of the reasons for the increase of phosphorylation of eNOS. Endothelial NO synthase activation can induce enhancement of cardiac angiogenesis. Indeed, activated VEGF/Akt/eNOS pathway, which occurs within 2 weeks from therapy origination, is vital to the proangiogenic effects (Rengo et al., 2012;Rengo et al., 2013). Previous study suggested that garcinia kola seed extract can activate Akt phosphorylation in an ischemic/reperfusion injury rat heart model (Oyagbemi et al., 2018). Besides, rusults in approximate agreement with previous reports, we also observed that GA increased the expression of p-Akt by verifying it from in vivo and in vitro experiments, as a consequence of which the downstream effectors eNOS is upregulated. Combined with recent other studies, we inferred that GA could be an effective pro-angiogenic drug in myocardial injury ultimately through Akt/eNOS activation.
In summary, the present study showed that GA treatment attenuated cardiac dysfunction and contributed to neovascularization in MI model through participating in the production of HIF-1α, VEGF and bFGF and the activation of p-Akt/eNOS. Thus, GA might be regarded as a candidate in the prevention and treatment of ischemic heart diseases.

Declarations
Ethics approval and consent to participate

Availability of data and materials
The data are not curated publicly and only available on permission of the approval of corresponding author.
Competing interests I declare that the aythors have no competing interests or other interests might be perceived to in uece the results and/or disccusion reperted in the paper.

Funding
This study was nancially supported by grants from the National Natural Science Foundation of China