Down-regulation CD36 increased proliferation of endothelial cell in hypertrophic cardiomyopathy

doi:10.21203/rs.3.rs-2890631/v1

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Down-regulation CD36 increased proliferation of endothelial cell in hypertrophic cardiomyopathy

https://doi.org/10.21203/rs.3.rs-2890631/v1

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Background: Hypertrophic cardiomyopathy (HCM) is a cardiovascular disease caused by genetic mutations. HCM with normal epicardial coronary arteries often reveal microvascular rarefaction and myocardial ischemia.Cluster of differentiation 36 (CD36) is a multifunctional pattern recognition membrane receptor that is highly expressed on microvascular endothelial cells (MVEC) and plays an essential role in endothelial cell function. However,whether CD36 affects vascular endothelial cell proliferation in HCM is unclear.We detected the effects of CD36 on endothelial cell proliferation and it’s underlying mechanisms in HCM.

Methods: We detected the effects of CD36 on endothelial cell proliferation and it’s underlying mechanisms in HCM. These studies were complemented by in vivo analysis of CD36 expression in cTnTQ92transgenic mice myocardium and in vitro analysis of transfection of CD36 gene or SiRNA-CD36 to endothelial cells isolated from cTnTQ92transgenic mice heart.

Results: In this study, we found that the expression of CD36 was higher and the microvessel density was lower in myocardial tissue of cTnTQ92 transgenic mice than that in the myocardial tissue of wild-type control C57BL/6 mice. The expressions of CD36 and p21 in endothelial cells isolated from cTnTQ92transgenic mice heart were higher than those in control group. The expression levels of proliferating cell nuclear antigen (PCNA) and Cyclin D1 proteins were lower than that of control group, and the proliferation of endothelial cells was lower than that of control group, but the opposite results were obtained by transfection of SiRNA-CD36 to endothelial cells. Further studies showed that down-regulation of CD36 increased the expression of p-VEGFR2 and increased proliferation of endothelial cell. EGFR specific inhibitor AG1478 reversed these effects.

Conclusions: These findings suggest down-regulation of CD36 increased the expression of p-VEGFR2 and increased proliferation of endothelial cell in hypertrophic cardiomyopathy. Down-regulation CD36 expression may be a potential therapeutic strategy for the treatment of myocardial ischemia in HCM.

Cluster of differentiation 36

Down-regulation

Endothelial growth factor receptor 2

Hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is a cardiovascular disease caused by genetic mutations, with a prevalence of approximately 0.20% in adults. HCM is mainly characterized by asymmetric ventricular wall hypertrophy and outflow tract obstruction, and is a common cause of sudden cardiac death in young patients[1]. Positron emission tomography (PET) revealed endocardial perfusion, coronary flow reserve and microvessel density was decreased and myocardial fibrosis was significantly increased in HCM myocardium with normal epicardial coronary arteries[2–4]. The above studies suggested that myocardium of HCM was in a hypoperfusion and ischemia. Myocardial ischemia is a risk factor for the development of heart failure in HCM and increases the risk of malignant arrhythmias and death[5, 6].

CD36 is a transmembrane protein, belonging to class B scavenger receptor family[7], which is widely expressed on the surface of myocardial microvascular endothelial cells. And it plays a key role in regulating endothelial cell function and proliferation[8]. Studies have shown that CD36 expression is significantly decreased on the surface of vascular endothelial cells in tumor tissues, and up-regulation of CD36 expression can significantly reduce the microvessel density of tumor tissues[9]. In HCM, whether regulation of CD36 expression levels can affect myocardial microvascular proliferation and improve myocardial ischemia has not yet been investigated.

The purpose of this study is to determine the effect of CD36 on HCM angiogenesis and to verify whether inhibition of CD36 can promote HCM myocardial endothelial cell proliferation.

Materials

Experimental animals: Cardiac troponin T (cTnTQ92) transgenic mice were provided by institute of laboratory animal sciences, CAMS&PUMC, China. Myocardial troponin T (cTnTQ92) transgenic mice, the animal model of HCM, was constructed by transferring cTnTQ92 into C57BL/6J. cTnTQ92 transgenic mice was characterized by reduced ventricular chamber, myocardial hypertrophy, myocyte disarray, interstitial fibrosis and diastolic dysfunction, which was an ideal animal model for the study of HCM. Ethyl carbamate was administered under general anesthesia by intraperitoneal injection.

Reagents and antibodies

CD7431 (Cat. No. DF7431, Affinity, China); HRP-labeled goat anti-rabbit IgG (Cat. No. 31460, thermoFisher, USA); VIII (Cat. No. AF0156, Affinity, China); CD36 antibody, (Cat. No.: sc-7309, Santa cruz, USA); cyclinD1 antibody (Cat. No.: WL01435a, wanleibio, China); PCNA antibody, (Cat. No.: WL03213, wanleibio, China); P21 antibody (Cat. No.: AF6290, Affinity, China); p-VEGFR2 antibody (Cat. No.: AF4426, Affinity, China); VEGR2 antibody (Cat. No.: WL02294, wanleibio, China); Fyn antibody (Cat. No.: WL04271, wanleibio, China); Secondary antibody Goat anti-rabbit IgG-HRP (wanleibio, China); Cell Proliferation Assay Kit (Cat. No.: KGA319, Kaiji Company, China); tsp1 antibody (Cat.No.: Abcam, UK)

Instrument

Electrophoresis apparatus (model: DYY-7C Beijing Liuyi Company, China); quantitative fluorescence PCR instrument (model: Exicycler 96, BIONEER Company, South Korea); flow cytometry (model: NovoCyte, Aceabio Company, USA); microplate reader (model: ELX-800, BIOTEK Company, USA); microscope (model BX53, OLYMPUS Company, Japan); microscope photography system (model: DP73, OLYMPUS Company, Japan).

MVEC isolation

Hearts harvested from cTNTQ92 transgenic mouse and wild-type control C57BL/6 mice were minced and enzymatically digested in MACS C-tube (Miltenyi Biotec). Cells were centrifuged, resuspended in PEB buffer (1x PBS, 0.5% BSA, 2mM EDTA), blocked with FcR blocking reagent (Miltenyi Biotec) then incubated with CD31 conjugated microbeads for 15 min at 4°C. The cell suspension was passed through MACS separation column, the column washed with PEB buffer (3X) and cells eluted and plated on gelatin-coated coverslips in 6 well plates.

Quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from cells and tissues using TRIzol reagent under RNase-free conditions, according to the manufacturer^, s instructions. All qualified RNA was reverse transcribed using random primers and stem-loop primers in the RT Reagent Kit system. β-Actin was used as the housekeeping gene. Relative expression was quantified using the formula 2-ΔΔCt. Specific primer sequences used for real-time PCR were listed in Table 1.

Table 1

qPCR Primer Sequences
Genes	Sequences
mus CD36	Forward: 5′-ACTGTGGGCTCATTGCT-3′ Reverse: 5′-CACGTCATCTGGGTTTT-3′
mus β-actin	Forward: 5′-CTGTGCCCATCTACGAGGGCTAT-3′ Reverse: 5′-TTTGATGTCACGCACGATTTCC-3′
homo CD36	Forward: 5′-CAGTTGGAGACCTGCTTA-3′ Reverse: 5′- AATGTTGCTGCTGTTCAT-3′
homo β-actin	Forward: 5′-GGCACCCAGCACAATGAA-3′ Reverse: 5′-TAGAAGCATTTGCGGTGG-3′

Western Blot Analysis

A 500 uL aliquot of cell lysate containing protease and phosphatase inhibitor was added to each well. After quantifying the total protein concentration by BCA protein kit, the lysates were added with dithiothreitol buffer at the same concentration and boiled for 5 min. The samples with the same protein concentration were determined by electrophoresis in SDS − polyacrylamide gel. The complete gel was then electroblotted onto an Immobilon-P membrane. The membrane was incubated in PBST buffer solution containing 5% BSA for 2 h, followed by incubation overnight with primary antibodies in PBS containing 1% BSA. Finally, after incubation with secondary antibodies (1:3000) in PBS buffer solution containing 1% BSA for 30 min, the bands of enhanced green fluorescent protein were detected by chemiluminescence. The cells after the treatment were assessed by Western blot similarly.

Gene silencing

Knockdown of CD36 in MVEC was achieved by using a reverse siRNA transfection procedure performed in six-well plates. Therefore, for each well to be transfected, 5ul Lipofectamine RNAiMAX (Life Technologies, Thermo Fisher Scientific, Eugene, Oregon, USA) were mixed with 500ul Opti-MEM (Gibco, Thermo Fisher Scientific, Eugene, Oregon, USA) and combined with 25 pmol siRNA (Genepharma, Shanghai, China). The transfection mixture was incubated at room temperature for 20min. HMVECs were harvested in complete growth medium without antibiotics and diluted so that 2ml contained the appropriate number of cells to give 30–50% confluence 24 hours after plating. Cell suspension were mixed with the transfection mixture and incubated. Replace the culture medium after 4–6 hours, and continue to culture for 24–48 hours.

Immunocytochemistry

Prior to treatment, heart tissue sections were washed in PBS, fixed for 15 minutes in precooled acetone, and washed thrice. Tissues were then permeabilized in 0.5% Triton X-100for 10 minutes and washed. Endogenous peroxidases were inactivated using 3% H2O2, followed by blocking with goat serum. Cells were incubated overnight at 4°first antibody. Then, Tissues were washed and incubated for 45 min with secondary antibody. Cytochemical reactions were performed using a diaminobenzidine kit and Tissues were counterstained with hematoxylin.

Bromodeoxyuridine (BrdU) staining

MVEC cells were added to 10uM BrdU and cultured in vitro at 37℃ for 45 min. Add 2mL flow cytometry staining buffer, centrifuge at room temperature 300-400xg for 5 min, and discard the supernatant. Add 100ul DNase I working solution and incubate at 37℃ for 1 hour away from light. 2mL flow cytometry staining buffer was added, centrifuged at room temperature 300-400xg for 5 min, and the supernatant was discarded. Then each sample was added with 5ul anti-BRDU fluorescent dye direct label antibody, mixed and incubated at room temperature for 20–30 min away from light. Proliferation of HUVEC cells was detected by flow cytometry.

Statistical processing

SPSS 17.0 statistical software was used for data analysis and processing. All measurement data were expressed as mean ± standard deviation, and t-test was used when there were only two groups of differences between groups, and one-way analysis of variance (one-way ANOVA) was used when there were multiple groups. P < 0.05 was considered statistically significant.

Microvascular Rarefaction in the myocardial tissue of cTNTQ92 transgenic mice

To assess the differences in myocardial microvascular density between cTNTQ92 transgenic mice and wild-type control C57BL/6 mice, microvessels were labeled with CD105 by immunohistochemistry (Fig. 1A-B). The result showed that microvessel density in the myocardial tissue of cTNTQ92 transgenic mice was significantly lower than that in the myocardial tissue of wild-type control C57BL/6 mice (Fig. 1B).

The level of CD36 expression was increased in myocardial tissue and endothelial cells isolated from cTnTQ92 transgenic mice heart.

To access the difference of CD36 expression in myocardial tissue between cTNTQ92 transgenic mice and the control group, the mRNA and protein expression levels of CD36 were analyzed by qRT-PCR and WT. The result showed that the expression of CD36 mRNA (Fig. 2A) and CD36 protein (Fig. 2B) in the myocardial tissue of cTNTQ92 transgenic mice were significantly higher than those in the control group, the same results (Fig. 3A-B) were seen in endothelial cells isolated from cTnTQ92 transgenic mice heart.

Endothelial cells proliferation in cTNTQ92 transgenic mice was decreased.

In order to access the difference of endothelial cells proliferation between cTNTQ92 transgenic mice and the control group, we culture endothelial cells isolated from mice heart for western blot analysis and bromodeoxyuridine (BrdU) staining. The result show that p21 (Fig. 3E) in endothelial cells of cTNTQ92 transgenic mice were higher than those of the control group by Western blot. However, the expression levels of PCNA (Fig. 3C) and Cyclin D1 (Fig. 3D) proteins were lower in cTNTQ92 transgenic mice than those of the control group. Meanwhile, BrdU staining and flow cytometry showed that the proliferation of endothelial cells in cTNTQ92 transgenic mice were lower than that in the control group (Fig. 3F, Fig. 4) .

Inhibition of CD36 expression promotes endothelial cells proliferation.

To access the effect of CD36 on endothelial cells proliferation, we transfected endothelial cells isolated from cTnTQ92 transgenic mice heart with siRNAs targeting CD36 (siRNA-CD36) to knockdown CD36 expression (Fig. 5C-D) and observed a decrease in cyclin-dependent kinase (CDK) inhibitor p21 protein (Fig. 5F). We also observed an increase in cyclin D1 (Fig. 5E) and PCNA protein (Fig. 5G) level after down-regulating the CD36 expression in endothelial cells, we also observed an increase in endothelial cell proliferation by BrdU staining and flow cytometry after down-regulating the CD36 expression in endothelial cells (Fig. 5B). Meanwhile, we constructed CD36 overexpressing (CD36 OE) stable cell lines in endothelial cells, finding that CD36 OE in endothelial cells reduced cyclin D1 (Fig. 5E) and PCNA protein (Fig. 5G) expression and increased p21 accumulation (Fig. 5F). We also observed an decrease in endothelial cells proliferation by BrdU staining and flow cytometry after up-regulating the CD36 expression in endothelial cells (Fig. 5B) .All of these data supported the notion that inhibition of CD36 expression promotes endothelial cells proliferation.

Down-regulation of CD36 increased the expression of p-VEGFR2 and increased proliferation of endothelial cells.

We next explored the underlying mechanism of decreased proliferation in CD36 OE endothelial cells. We found that down-regulation of CD36 by SiRNA-CD36 increased the expression of phosphorylated vascular endothelial growth factor receptor2 (p-VEGFR2) (Fig. 6B) and the proliferation of endothelial cells (Fig. 6D). However, AG1478, a specific inhibitor of EGFR, suppressed the expression of p-VEGFR2 (Fig. 6B) and the proliferation of endothelial cells (Fig. 6D), indicating that down-regulation of CD36 enhances phosphorylation of pro-angiogenic VEGFR, and increases the proliferation of endothelial cells.

Myocardial ischemia of HCM accelerates the progression of heart failure and increases the risk of malignant arrhythmias and sudden cardiac death. CD36 is widely expressed on the surface of myocardial microvascular endothelial cells and plays a key role in regulating endothelial cell function and proliferation. In this study, we found that down-regulation of CD36 increased the expression of p-VEGFR2 and increased proliferation of endothelial cell in hypertrophic cardiomyopathy. Down-regulation CD36 expression may be a potential therapeutic strategy for the treatment of myocardial ischemia.

In this study, we found that the microvessel density in myocardium of cTNTQ92 transgenic mice was significantly lower. This observations is consistent with our previous findings for human HCM myocardium [4]. We also found the expression levels of both CD36 mRNA and CD36 protein in the myocardium of cTNTQ92 transgenic mice were significantly higher than those of the control group, which suggest that CD36 expression levels may be associated with angiogenesis in HCM. p21 is a traditional cell cycle inhibitor, which acts as a cell cycle arrest through interference with a variety of genes involved in deoxyribose replication and cell division[10]. PCNA is present in the nucleus and is an accessory protein of DNA polymerase δ, whose concentration changes periodically during the cell cycle. It can be used as an indicator of cell proliferation status [11]. Cyclin D1 promotes cell proliferation by combining with and activating CDK4, a cyclin-dependent kinase unique to G1 phase [12]. Our findings showed that p21 in endothelial cells isolated from cTNTQ92 transgenic mice heart were higher than those of the control group by Western blot. However, the expression levels of PCNA and Cyclin D1 proteins were lower in cTNTQ92 transgenic mice than those of the control group. Meanwhile, BrdU staining and flow cytometry showed that the proliferation of endothelial cells in cTNTQ92 transgenic mice were lower than that in the control group. The above findings suggesting that CD36, which is highly expressed in myocardium and vascular endothelial cell of cTNTQ92 transgenic mice, may affect the angiogenesis of vascular endothelial cells. To access the effect of CD36 on endothelial cells proliferation, we transfected endothelial cells with siRNAs targeting CD36 (siRNA-CD36) or cDNA of CD36 to regulate CD36 expression. We observed a decrease in cyclin-dependent kinase inhibitor p21 protein and an increase in cyclin D1 and PCNA protein level after down-regulating the CD36 expression in endothelial cells, we also observed an increase in endothelial cells proliferation after down-regulating the CD36 expression, but the opposite results were obtained by CD36 overexpression. These studies suggest that inhibition of CD36 expression by siRNA-CD36 promoted endothelial cell division and increases endothelial cell proliferation. These observations is consistent with the report that reduction of CD36 expression increases endothelial cell proliferation[13, 14]. The mechanism of decreased proliferation in CD36 OE endothelial cells is probably multifactorial. In endothelial cells, vascular endothelial growth factor receptor 2 (VEGFR2) recognizes its ligand VEGF and becomes phosphorylated. VEGFR2 than transduces signals to mediate cellular migration, proliferation, and tube formation important for angiogenesis[15, 16]. Our results showed down-regulation of CD36 increased the expression of p-VEGFR2 and increased proliferation of endothelial cell. EGFR specific inhibitor AG1478 reversed these effects. And other studies found that CD36 decreased VEGFR phosphorylation levels after combining with TSP1, similar to the results of this study[17].

Limitations

The limitation of this study is that overexpression of CD36 could significantly inhibit the proliferation of endothelial cells and failed to eliminate the effect of hypertrophic cardiomyocytes on endothelial cell proliferation. In addition, whether CD36 has an effect on cardiomyocyte hypertrophy was not further investigated in this study, and the effect of CD36 on cardiomyocyte hypertrophy could not be clarified. Future studies could focus on the effect of CD36 on the degree of cardiomyocyte hypertrophy for in-depth study.

In summary, These findings suggest that CD36 plays a negative role in angiogenesis in HCM, down-regulation CD36 expression can be a potential therapeutic strategy for the treatment of myocardial ischemia in HCM.

Cluster of differentiation 36 (CD36), Proliferating cell nuclear antigen (PCNA), siRNAs targeting CD36 (siCD36), CD36 overexpressing (CD36 OE), siRNAs targeting negative control (siRNA-NC), wild-type mice (WT mice), lentivirus empty vector (Vector), quantitative real-time PCR (qRT-PCR), hypertrophic cardiomyopathy (HCM), endothelial growth factor receptor 2 (VEGFR2), vascular endothelial growth factor receptor 2 (VEGFR2), microvascular endothelial cells (MVEC), cardiac troponin T transgenic mouse (cTNTQ92), Positron emission tomography (PET), cyclin-dependent kinase (CDK), Bromodeoxyuridine (BrdU).

Acknowledgements

We thank institute of laboratory animal sciences, CAMS&PUMC, China for providing the cTNTQ92 transgenic mice.

Funding

This research was supported by the Natural Science Foundation of Ningxia, china (2020AAC02034), special talent start - up program of ningxia medical university (XZ20200012) .

Availability of data and materials

The datasets used during the current study available from the corresponding author on reasonable request.

Authors’ contributions

HWT, LJG, CZY, and QY contributed to the design of the study. HWT, XHW and LNM contributed to the analysis, while all authors (HWT, QY, XHW, LNM, YX, YW, XHM, SJX, LJG) contributed to the interpretation of data. HWT drafted the manuscript and QY, YX, and LJG contributed significantly to the preparation. All the authors critically revised the manuscript and gave final approval and agree to be accountable for all aspects of the work, ensuring both its integrity and accuracy.

Ethics approval and consent to participate

All experimental protocols were approved by the Ethics Committee of People's Hospital of Ningxia Hui Autonomous Region, all methods were carried out in accordance with relevant guidelines and regulations. This study was carried out in compliance with the ARRIVE guidelines.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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No competing interests reported.

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Down-regulation CD36 increased proliferation of endothelial cell in hypertrophic cardiomyopathy

Status:

Version 1

Abstract

Figures

Introduction

Methods

Materials

Reagents and antibodies

Instrument

MVEC isolation

Quantitative real-time PCR (qRT-PCR)

Western Blot Analysis

Gene silencing

Immunocytochemistry

Bromodeoxyuridine (BrdU) staining

Statistical processing

Results

Discussion

Limitations

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1