HSPA12A Stimulates p38/ERK-AP-1 Signaling to Promote Angiogenesis and Is Required for Functional Recovery Postmyocardial Infarction

Li, Tingting; Wu, Jun; Yu, Wansu; Mao, Qian; Cheng, Hao; Zhang, Xiaojin; Li, Yuehua; Li, Chuanfu; Ding, Zhengnian; Liu, Li

doi:https://doi.org/10.1155/2022/2333848

Oxidative Medicine and Cellular Longevity

On this page

Abstract Introduction Materials and Methods Results Discussion Conclusion Data Availability Conflicts of Interest Authors’ Contributions Acknowledgments Supplementary Materials References Copyright Related Articles

Research Article | Open Access

Volume 2022 | Article ID 2333848 | https://doi.org/10.1155/2022/2333848

HSPA12A Stimulates p38/ERK-AP-1 Signaling to Promote Angiogenesis and Is Required for Functional Recovery Postmyocardial Infarction

Tingting Li,¹Jun Wu,¹Wansu Yu,¹Qian Mao,²Hao Cheng,³Xiaojin Zhang,¹Yuehua Li,⁴Chuanfu Li,⁵Zhengnian Ding,²and Li Liu^1,4

Academic Editor: Yong Zhou

Received29 Nov 2021

Revised18 May 2022

Accepted26 May 2022

Published22 Jun 2022

Abstract

Angiogenesis plays a critical role in wound healing postmyocardial infarction (MI). However, there is still a lack of ideal angiogenic therapeutics for rescuing ischemic hearts clinically, suggesting that a more understanding regarding angiogenesis regulation is urgently needed. Heat shock protein A12A (HSPA12A) is an atypical member of the HSP70 family. Here, we demonstrated that HSPA12A was upregulated during endothelial tube formation, a characteristic of in vitro angiogenesis. Intriguingly, overexpression of HSPA12A promoted in vitro angiogenic characteristics including proliferation, migration, and tube formation of endothelial cells. By contrast, deficiency of HSPA12A impaired myocardial angiogenesis and worsened cardiac dysfunction post-MI in mice. The expression of genes related to angiogenesis (VEGF, VEGFR2, and Ang-1) was decreased by HSPA12A deficiency in MI hearts of mice, whereas their expression was increased by HSPA12A overexpression in endothelial cells. HSPA12A overexpression in endothelial cells increased phosphorylation levels and nuclear localization of AP-1, a transcription factor dominating angiogenic gene expression. Also, HSPA12A increased p38 and ERK phosphorylation levels, whereas inhibition of p38 or ERKs diminished the HSPA12A-promoted AP-1 phosphorylation and nuclear localization, as well as VEGF and VEGFR2 expression in endothelial cells. Notably, inhibition of either p38 or ERKs diminished the HSPA12A-promoted in vitro angiogenesis characteristics. The findings identified HSPA12A as a novel angiogenesis activator, and HSPA12A might represent a viable strategy for the management of myocardial healing in patients with ischemic heart diseases.

1. Introduction

Myocardial infarction (MI) is the irreversible death of cardiac cells secondary to prolonged ischemia and can transit to heart failure which accounts for one of the leading causes of death worldwide [1, 2]. Angiogenesis, the new blood vessel formation, has been shown to have the potential to rescue ischemic myocardia early after MI and also is critical to prevent long-term cardiac remodeling to prevent heart failure development [3–5]. Therefore, managing angiogenesis to timely and effectively restore blood flow is a promising therapeutic approach for healing MI hearts.

Angiogenesis is a complex process initiated by the proliferation and migration of endothelial cells (ECs) in response to angiogenic factors. Among these angiogenic factors, the vascular endothelial growth factor (VEGF) is considered especially critical due to its powerful role in the activation of EC proliferation and migration by binding to its receptors such as VEGFR2 [6, 7]. VEGF is also helpful for EC survival under ischemic conditions. In addition to the VEGF-dependent pathway, the Angiopoietin- (Ang-) mediated signaling is necessary for angiogenesis through promoting EC proliferation and migration, maturing new blood vessels, and maintaining vessel stability [8, 9]. Accordingly, inhibitors targeting VEGF- or Ang-dependent pathways suppress solid tumor growth clinically or preclinically and also are effective for age-related macular degeneration treatment [10, 11]. Specifically, several clinical trials for evaluating therapeutic angiogenesis after MI have been performed. For example, EPICCURE is an ongoing clinical trial by delivering VEGF mRNA to ischemic but viable myocardia to assay its safety and potential angiogenic effects [12]. However, a more comprehensive understanding regarding angiogenesis regulation is still needed.

Activator protein-1 (AP-1) is a protein dimer formed by Jun, Fos, or ATF and emerged as a critical transcription factor for angiogenesis at both the basal and inducible levels through controlling VEGF and other angiogenic gene expressions [13]. Following phosphorylated by kinases such as MAPK (ERKs, p38, and JNKs) and PI3K/Akt signaling, AP-1 translocates to nuclei to drive target gene expression [14, 15]. Therefore, activating AP-1-mediated angiogenic signaling might be beneficial for MI repairment.

Heat shock protein A12A (HSPA12A) was identified in 2003 and classed as a distant member of the heat shock protein 70 family [16]. Subsequent studies revealed the downregulation of HSPA12A in the brains of patients with schizophrenia [17]. Recently, we have reported that HSPA12A is necessary for cerebral protection from stroke, for development of obesity and nonalcohol liver disease, and for growth of liver cancer [18–21]. Particularly, we found that HSPA12A facilitates angiogenesis in mouse liver carcinoma [21] and increases basal levels of ERK phosphorylation and VEGF expression in endothelial cells [21, 22]. Therefore, it is possible that HSPA12A might promote angiogenesis to attenuate post-MI cardiac dysfunction.

To test this possibility, we examined the effects of HSPA12A on angiogenesis and post-MI cardiac performance. We found that HSPA12A was upregulated during endothelial tube formation, while the knockout of HSPA12A in mice impaired cardiac angiogenesis and worsened cardiac dysfunction post-MI. Further molecular analysis revealed that the HSPA12A-promoted angiogenesis was mediated by activating the p38/ERK-AP-1 signaling axis. The findings indicate that HSPA12A may represent a potential target for cardioprotection against ischemia through promoting angiogenesis.

2. Materials and Methods

2.1. Reagents and Antibodies

The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reagent and primary antibodies for α-smooth muscle actin (α-SMA) and α-tubulin were purchased from Sigma-Aldrich (St. Louis, MO). The primary antibody for GAPDH was from Bioworld Technology (St. Louis Park, MN). Primary antibodies for c-Jun, phosphor-c-Jun (p-c-Jun), p38, phosphor-p38, ERKs, phosphor-ERKs (p-ERKs), JNKs, and phosphor-JNKs (p-JNKs) were purchased from Cell Signaling Technology (Beverly, MA). The primary antibody for Ang-1 was from Santa-Cruz Biotechnology (Dallas, TX). The primary antibody for VEGFR2 was from SAB (Baltimore, MD). Primary antibodies for HSPA12A and CD31 were from Abcam (Cambridge, MA). The primary antibody for VEGF was purchased from Millipore (Billerica, MA). Matrigel was a product from BD Biosciences (San Jose, CA). Bovine serum albumin (BSA) was a product from Roche (Basel, Switzerland). Normal goat serum was a product of Jackson Immuno Research (West Grove, PA). Fetal bovine serum (FBS) and the M199 medium were products from Biological Industries (Kibbutz Beit HaEmek, Israel). PD98059 and SB203580 were from MedChemExpress (Monmouth Junction, NJ).

2.2. Animals

Conditional Hspa12a knockout mice (Hspa12a^-/-) were generated using the loxP and Cre recombinant system as described in our previous studies [19, 20, 23]. Briefly, the region of the Hspa12a gene containing exons 2–4 was retrieved from a 129/sv BAC clone (BAC/PAC Resources Center, Oakland, CA) using a retrieval vector containing two homologous arms. Exons 2 and 3 were replaced by loxP sites flanking a PGK-neo cassette as a positive selection marker. Embryonic stem cells were electroporated with the linearized targeting vector, selected, and then expanded for Southern blotting analysis. Chimeric mice (Hspa12a^flox/+) were generated by injecting embryonic stem cells into C57BL/6 blastocysts, followed by transfer into pseudopregnant mice. To remove the Hspa12a gene, the chimeric mice were crossed with EIIa-Cre transgenic mice. The mice were bred at the Model Animal Research Center of Nanjing University and were maintained in the Animal Laboratory Resource Facility of the same institution. The animal care and experimental protocols were approved by Nanjing University’s Committee on Animal Care. All experiments conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication, 8th Edition, 2011).

Mice (C57BL/6 strain) were randomly assigned to all analyses. Investigators were blinded to the histological analysis. Investigators involved in animal handling, sampling, and raw data collection were not blinded.

2.3. Myocardial Infarction (MI) Surgery in Mice

MI was induced in 8- to 12-week-old male Hspa12a^-/- mice and their wild-type (WT) littermates by permanently ligating the left anterior descending coronary (LAD) according to our previous methods [2, 3]. Briefly, mice were anesthetized by inhalation of 1.5–2% isoflurane. The adequacy of anesthesia was assayed by the disappearance of righting reflex and pedal withdrawal reflex. After anesthesia, mice were subjected to mechanical ventilation, chest opening along the left sternal border, and permanent LAD ligation. For sham operation, the same surgery was conducted except for the LAD occlusion. Body temperature was maintained at 36.8–37.1°C using a heating platform throughout the surgical procedure. For tissue collection, mice were sacrificed by overdose anesthesia (pentobarbital sodium 150 mg/kg intraperitoneal injection) and cervical dislocation.

2.4. Echocardiographic Measurement in Mice

Two-dimensional echocardiography was performed to examine cardiac function using the Vevo770 system equipped with a 35 MHz transducer (VisualSonics, Toronto, Canada) according to our previous methods [2, 3, 24]. Briefly, after being anesthetized by inhalation with 1.2% isoflurane, mice were laid on the equipped plate and chest hair was shaved. Subsequently, the measurements were performed by an observer blinded to the treatment. The parameters were obtained in the M-mode tracings at the papillary muscle level and averaged using three to five cardiac cycles. Ejection fraction (EF%) and fraction shortening (FS%) of left ventricles were used to indicate cardiac systolic function.

2.5. Primary Human Umbilical Vein Endothelial Cell (HUVEC) Isolation and Growth

HUVECs were obtained from the umbilical vein cords of normal pregnancies according to our previous methods [22, 25]. Briefly, endothelial cells were dissociated from umbilical veins with 0.25% trypsin and grown in the M199 medium supplemented with 10% FBS and 0.5 ng/ml bFGF. The HUVECs in passages 2 to 5 were used in the experiments. The studies were approved by the Ethical Board of the First Affiliated Hospital of Nanjing Medical University (#2021-SR-104). All the human study procedures were followed in accordance with the ethical standards of the responsible committee on human experimentation and with the Helsinki Declaration of 1975, as revised in 2000. Human pulmonary artery endothelial cells (HPAEC) were obtained from Sciencell Research Laboratories (Carlsbad, CA, USA) and grown in endothelial cell medium-2 (ECM-2) (Sciencell) with 5% FBS and 1% endothelial cell growth factors according to previous methods [26].

2.6. Overexpression of HSPA12A in HUVECs

To overexpress HSPA12A (Hspa12a^o/e) in HUVECs, cells were infected with adenovirus that carrying the Hspa12a expression coding sequence as our previous methods [19, 20, 23]. The adenoviral vector containing the 3 Flag-tagged mouse Hspa12a coding region (NM_175199) was generated by GeneChem Company (Shanghai, China). The scheme of virus construction is shown in Figure S1. The cells infected with empty adenovirus served as normal controls (NC).

For p38 or ERK inhibition, SB203580 (20 μM) or PD98059 (25 μM) were introduced to cell cultures 24 h after HSPA12A overexpression.

2.7. Matrigel-Based In Vitro Angiogenesis Assay

In vitro endothelial angiogenesis was assessed by tube formation using Matrigel-based assay [27]. Briefly, after HSPA12A overexpression for 24 h, HUVECs ( cells/well) were seeded on growth factor-reduced Matrigel-coated 96-well plates. Cells were photographed at 2.5 and 4 h after being grown on Matrigel using a microscope at a magnification of 40x. Tube formation was expressed as total branch length/field and master junctions/field using NIH ImageJ software.

2.8. Endothelial Cell Migration Assay

The migration capacity of HUVECs was measured by the scratch (or wound healing) assay according to our previous methods [28]. Briefly, HUVECs grown in 6-well plates were scratched with 200 μl tips when cells reached 80% confluence. After scratching, cells were incubated with a 3% FBS-supplemented M199 medium and photographed at the indicated time points. The percentage of wound closure was analyzed by an image analyzer (NIH ImageJ software).

2.9. MTT Assay

MTT assays were performed to evaluate the viability of HUVECs after overexpression of HSPA12A for 24, 48, and 72 h according to our previous methods [21]. In brief, HUVECs were incubated with MTT (0.5 mg/ml) for 4 h. The crystals that formed were solubilized in DMSO, and the color was read on a Synergy HT plate reader at 570 nm (Synergy HT, BioTek, USA).

2.10. Western Blotting Analysis

Cellular proteins were prepared from infarcts of mice 7 days after MI or from HUVECs 24 h after HSPA12A overexpression [18]. Briefly, after separating by SDS-PAGE and transferring onto Immobilon-P membranes, the membranes were incubated with the primary antibodies and subsequently incubated with peroxidase-conjugated secondary antibodies. After detection with a chemiluminescent substrate, the signals were captured using scanning densitometry. Results from each set of experiments were presented as the relative integrated intensities (compared to those of controls).

2.11. Immunofluorescence Staining

For analyzing myocardial angiogenesis, cardiac tissues were collected transversely at papillary muscles for immunofluorescence staining according to our previous methods [2, 3, 29]. Briefly, the tissues were prepared for cryosectioning at a thickness of 4 μm. After blocking with 7.5% normal goat serum (1 hour), tissue sections were probed with the primary antibody (4°C, overnight) and followed by incubation with secondary antibodies to visualize the staining. The DAPI reagent was used to counterstain nuclei. The staining was observed, captured, and quantified using a fluorescence microscope (Olympus, Japan).

For immunocytochemistry in primary endothelial cells, cells grown on coverslips were fixed with acetone/methanol (1 : 1) and followed by immunostaining as mentioned above.

2.12. EdU Incorporation Assay

EdU incorporation assay was performed to indicate cell proliferation according to our previous methods [21]. After overexpression of HSPA12A for 48 h, HUVECs were incubated with EdU (50 μM, 2 h, 37°C). After fixation with 4% formaldehyde (30 min), cells were permeabilized with 0.5% Triton X-100 (room temperature, 10 min), followed by incubation with Apollo reaction cocktail (30 min). DAPI was used to counterstain nuclei. The proportion of cells that incorporated EdU was determined by fluorescence microscopy.

2.13. Histological Examination

Following MI for 14 days, cardiac tissues at papillary muscle levels were collected and fixed with 4% paraformaldehyde for 24 h. After paraffin-embedded sectioning was prepared, hematoxylin-eosin (HE) staining was performed. The staining was observed under a microscope.

2.14. Statistics

Results are presented as the . Results were analyzed by Student’s two-tailed unpaired -test, one-way analysis of variance (ANOVA), or two-way analysis of variance, followed by a Tukey test as a post hoc test. was considered statistically significant.

3. Results

3.1. HSPA12A Is Upregulated during Endothelial Tube Formation

To investigate whether HSPA12A is involved in angiogenesis, an in vitro experiment was performed to examine HSPA12A expression in endothelial cells during tube formation. To this end, HUVECs grown on Matrigel-coated plates were subjected to hypoxia to induce tube formation according to previous studies [30]. Hypoxia was achieved by incubation of HUVECs with a medium containing 3% FBS in an incubator containing 1% O₂, 94% N₂, and 5% CO₂ for 6 h (Figure 1(a)). We found that hypoxia induced tube formation, as indicated by increases of both total branch length and master junction numbers (Figure 1(b)). Notably, HSPA12A displayed a 2-fold higher expression during hypoxia-induced tube formation than normoxia HUVECs (Figure 1(c)).

(a) Experimental protocol

(b) Tube formation. Hypoxia-induced tube formation was examined by Matrigel assay. , /group.

(c) HSPA12A protein expression. HSPA12A protein expression was analyzed by immunoblotting in HUVECs after hypoxia or normoxia treatment. , /group

3.2. HSPA12A Promotes Proliferation of HUVECs

The upregulation of HSPA12A in endothelial cells during tube formation prompts us to elucidate whether HSPA12A plays a role in angiogenesis. To this end, HSPA12A was overexpressed (Hspa12a^o/e) in HUVECs by infection with Hspa12a-adenovirus, and cells infected with empty adenovirus served as normal controls (NC) (Figures 2(a) and 2(b), Figure S2). Endothelial proliferation is a key step for angiogenesis [31]. MTT assay showed higher viability in Hspa12a^o/e HUVECs after HSPA12A overexpression for 72 h than the time-matched NC group (Figure 2(c)). This finding was confirmed by cellular morphological examination (Figure 2(d)). Moreover, Hspa12a^o/e HUVECs displayed more EdU incorporation than NC (Figure 2(e)). Together, the data indicate that endothelial proliferation is promoted by HSPA12A.

(a) Experimental protocol

(b) Overexpression of HSPA12A in HUVECs. Overexpression of HSPA12A in Hspa12ao/e HUVECs was confirmed by immunoblotting

(c) Cell viability. MTT assays were performed to examine cell viability after HSPA12A overexpression for 24, 48, and 72 h. , for 24 and 48 h groups and for 48 h groups

(d) Cell density observation. Phase-contrast microscope was used to examine cell density at the indicated times after HSPA12A overexpression.

(e) EdU incorporation assay. Cell proliferation was examined by EdU incorporation after overexpression of HSPA12A for 48 h. , /group.

3.3. HSPA12A Promotes Motility and Tube Formation of HUVECs

Motility and tube formation of endothelial cells are essential for angiogenesis. To examine the effect of HSPA12A on endothelial motile capacity, wound healing assay was performed as we described recently [28] (Figure 3(a)). We found that overexpression of HSPA12A in HUVECs promoted the migration rate after wounding for 6, 12, and 24 h, respectively, compared to the time-matched NC group (Figure 3(b)). Moreover, overexpression of HSPA12A in HPAEC promoted the migration capacity in the wound healing assay (Figure S3). Next, tube formation was examined using Matrigel-based assay [27]. Hspa12a^o/e HUVECs displayed significantly longer branch length after growing on Matrigel for 2.5 and 4 h than the time-matched NC group (Figure 3(c)). Similarly, significantly more master junctions were demonstrated in Hspa12a^o/e HUVECs after growing on Matrigel than in the time-matched NC group (Figure 3(c)).

(a) Experimental protocol

(b) Wound healing assay. Cell migration was examined by wound healing assay after wounding for 6, 12, and 24 h. vs. the time-matched NC. /group.

(c) Tube formation. Angiogenesis was examined after being grown on Matrigel for 2.5 and 4 h. and vs. the time-matched NC, /group.

3.4. HSPA12A Upregulates Angiogenic Gene Expression in HUVECs

Angiogenesis is strongly regulated by VEGF signaling through VEGF receptors such as VFGFR2 [32]. Overexpression of HSPA12A in HUVECs for 24 h significantly increased VEGF expression compared to the NC group (Figures 4(a) and 4(b)). Similar results were found in VEGFR2 examination, which showed markedly higher expression in Hspa12a^o/e HUVECs than in the NC group (Figure 4(b)). Angiopoietin-1 (Ang-1) is also important for angiogenesis through promoting endothelial proliferation and migration, maturing new blood vessels, and maintaining vessel stability [33–35]. Intriguingly, significantly higher Ang-1 protein expression was found in Hspa12a^o/e HUVECs compared to the NC group (Figure 4(b)).

(a) Experimental protocol

(b) Protein expression. Immunoblotting was performed to examine the indicated protein expression after HSPA12A overexpression for 24 h. , -9/group

3.5. HSPA12A Increases c-Jun/AP-1 Phosphorylation and Nuclear Localization in HUVECs

The expression of angiogenic genes, such as VEGF, is regulated by several transcriptional factors including AP-1 and Hif-1α [36, 37]. No difference in Hif-1α expression levels was found between the Hspa12a^o/e and NC HUVEC groups (Figures 5(a) and 5(b)). However, c-Jun, a component of AP-1 dimers, displayed significantly higher phosphorylation levels in Hspa12a^o/e HUVECs than in the NC group (Figure 5(b)). Accordingly, more nuclear content of c-Jun/AP-1 was found in Hspa12a^o/e HUVECs than in NC (Figure 5(c)). Moreover, in the Ad-HSPA12A group, the HUVECs with stronger HSPA12A staining also displayed stronger c-Jun staining in nuclei (Figure S4).

(a) Experimental protocol

(b) Protein expression. Immunoblotting was performed to examine the indicated protein expression after HSPA12A overexpression for 24 h. , /group

(c) Nuclear c-Jun/AP-1 fluorescence. Nuclear localization of c-Jun/AP-1 in HUVECs was examined by immunofluorescence staining. , /group.

3.6. HSPA12A Increases p38 and ERK Phosphorylation Levels

The phosphorylation of c-Jun/AP-1 is critical for its translocation to nuclei, where it exerts a transcription role to drive target gene expression. Considering that c-Jun can be phosphorylated by MAPKs and Akt [14, 15, 38], we examined the effects of HSPA12A on phosphorylation levels of MAPKs (p38, ERKs, and JNKs) and Akt (Figure 6(a)). No difference in phosphorylation levels of JNKs and Akt was found between Hspa12a^o/e and NC (Figure 6(b)). However, Hspa12a^o/e HUVECs displayed significantly higher phosphorylation levels of p38 and ERKs than NC (Figure 6(b)).

(a) Experimental protocol

(b) Immunoblotting. Protein expression and phosphorylation of the indicated genes were examined by immunoblotting after HSPA12A overexpression for 24 h. and , -6/group

3.7. Inhibition of p38 Attenuated the HSPA12A-Promoted c-Jun/AP-1 Phosphorylation, Angiogenic Gene Expression, and In Vitro Angiogenic Phenotypes of HUVECs

To determine the roles of p38 activation in HSPA12A-promoted in vitro angiogenesis, HUVECs were treated with p38 inhibitor SB203580 for 2 h (Figure 7(a)). Intriguingly, SB203580 removed the HSPA12A-induced increase of c-Jun/AP-1 phosphorylation (Figure 7(b)). The upregulation of VEGF and VEGFR2 by HSPA12A overexpression was also abolished by SB203580 (Figure 7(c)). Notably, HSPA12A-induced promotion of proliferation of HUVECs was diminished by SB203580, as evidenced by both MTT and morphological examination (Figures 8(a) and 8(b)). Moreover, HSPA12A-induced promotion of migration ability and tube formation capacity was removed by SB203580 (Figures 8(c) and 8(d)), suggesting that the HSPA12A-promoted angiogenesis is p38 dependent.

(a) Experimental protocol

(b) Phosphorylation of p38 and c-Jun/AP-1. Immunoblotting was performed to examine p38 and c-Jun/AP-1 phosphorylation levels following treatment with p38 inhibitor SB203580 for 2 h. and , for p38 groups and for c-Jun/AP-1 groups

(c) Expression of angiogenic genes. Immunoblotting was performed to examine the expression of the indicated genes following treatment with p38 inhibitor SB203580 for 2 h. , for the (SB203580+Hspa12a^o/e) VEGFR2 group and for all the other groups

(a) Cell viability. MTT assays were performed to examine cell viability. vs. the time-matched SB203580+Hspa12a^o/e group. /group

(b) Cellular morphological examination. Phase-contrast microscope was used to examine cellular morphology to illustrate cell density. /group.

(c) Wound healing assay. Migration was examined by wound healing assay. vs. the time-matched SB203580+Hspa12a^o/e group, /group.

(d) Tube formation. Tube formation was examined based on Matrigel assay. vs. the time-matched SB203580+Hspa12a^o/e group, /group.

Considering that ERK phosphorylation was also increased by HSPA12A, we therefore investigated the roles of ERKs in HSPA12A-promoted angiogenesis. To this end, HUVECs were treated with ERK inhibitor PD98059 (Figure 9(a)). ERK inhibition with PD98059 attenuated the HSPA12A-induced increases of c-Jun/AP-1 phosphorylation of VEGF and VEGFR2 expression (Figures 9(b) and 9(c)). Though inhibition of ERKs did not impact the HSPA12A-promoted cell proliferation, the HSPA12A-promoted migration and tube formation were attenuated following PD98059 treatment (Figures 10(a)–10(c)).

(a) Experimental protocol

(b) Phosphorylation of ERKs and c-Jun/AP-1. Immunoblotting was performed to examine ERKs and c-Jun/AP-1 phosphorylation levels following treatment with ERK inhibitor PD98059 for 2 h. , for ERK groups and for c-Jun/AP-1 groups

(c) Expression of angiogenic genes. Immunoblotting was performed to examine the expression of the indicated genes following treatment with ERK inhibitor PD98059 for 2 h. , for the (PD98059+Hspa12a^o/e) VEGFR2 group and for all the other groups

(a) Cell viability. MTT assays were performed to examine cell viability. /group

(b) Migration assay. Migration was examined by wound healing assay. vs. the time-matched PD98059+Hspa12a^o/e group, /group.

(c) Tube formation. Tube formation was examined based on Matrigel assay. vs. the time-matched PD98059+Hspa12a^o/e group, /group.

3.8. Deficiency of HSPA12A in Mice Impaired Angiogenesis and Worsened Cardiac Dysfunction after MI in Mice

Finally, we were interested to know the biological roles of HSPA12A in post-MI angiogenesis and cardiac performance in intact animals. To this aim, mice with the HSPA12A knockout (Hspa12a^-/-) and their gender-matched wild-type (WT) littermates were subjected to MI or sham surgeries (Figure 11(a)). The successful deletion of HSPA12A expression in Hspa12a^-/- hearts is shown in Figure 11(b). Angiogenesis that was examined by immunostaining for CD31 (PECAM-1) demonstrated that though no significant difference in capillary density was found between genotypes at basal levels (Figure S5), a lower capillary density was found in infarct regions of Hspa12a^-/- mice than in WT controls after MI for 14 days (Figure 11(c)). In supporting this, the hearts of Hspa12a^-/- mice displayed lower expression levels of VEGF and Ang-1 than the hearts of WT mice following MI (Figure 11(d)). In line with the reduced capillary density, Hspa12a^-/- mice demonstrated worsened cardiac dysfunction as indicated by lower left ventricular ejection fraction (EF%) and fraction shortening (FS%) than WT mice after MI for 14 days (Figure 11(e)). Additionally, histological examination by HE staining on paraffin-embedded sections, which were cut at left ventricular papillary muscle levels, showed a thinner free wall of the left ventricle in Hspa12a^-/- mice than WT controls after MI for 14 days (Figure S6). Altogether, deficiency of HSPA12A in mice impaired post-MI angiogenesis and cardiac function.

(a) Experimental protocol

(b) Deletion of HSPA12A in Hspa12a-/- hearts. HSPA12A protein expression was examined in the hearts of mice using immunoblotting. Note that HSPA12A was absent in Hspa12a-/- hearts

(c) Capillary density. Ventricular tissues at the papillary muscle level were collected after MI for 14 days. Cryosections were prepared for immunofluorescence staining against CD31 to illustrate capillary density. The representative images were from three independent experiments. , /group.

(d) Angiogenic gene expression. Infarcts of mouse hearts were collected to examine the expression of the indicated genes immunoblotting after MI for 7 days. , /group

(e) Cardiac function. Cardiac function was examined 14 days after MI using two-D echocardiography. The representative M-mode images at the papillary muscles are shown in the left panel. Parameters were obtained from M-mode tracings. vs. the time-matched WT mice, -11/group

4. Discussion

The major finding of this study is that HSPA12A was upregulated during endothelial tube formation, and overexpression of HSPA12A increased proliferation, migration, and tube formation in endothelial cells. By contrast, the knockout of HSPA12A impaired angiogenesis and worsened cardiac dysfunction post-MI in mice. Further analysis revealed that HSPA12A promoted angiogenesis through activating AP-1 in p38- and ERK-dependent mechanisms. The findings identified HSPA12A as a novel angiogenic regulator and suggest that HSPA12A might serve as an alternative approach for the management of angiogenesis-related diseases such as myocardial infarction.

Heat shock proteins (HSPs) were originally identified as stress-responsive proteins required for cell survival during thermal stress, acting as molecular chaperones [39]. Now, it is clear that HSPs can respond to a wider variety of insults such as oxidants, heavy metals, and hypoxia/ischemia. According to their molecular size, HSPs are currently classified into structurally unrelated subfamilies, including HSPA/HSP70, HSPB/HSP27, HSPC/HSP90, HSPH/HSP110, and NDAJ/HSP40 [28, 40]. Several HSPs, especially HSP90, HSP70-1A, and HSP27, have been reported to stimulate angiogenesis to promote tumor growth [41, 42]. In hearts, HSP20 attenuates diabetic cardiomyopathy through improving angiogenesis in mice [43]. Intriguingly, we have demonstrated that HSPB1/HSP27 and HSPA12B are required for angiogenesis and cardiac functional recovery after MI in mice [2, 3]. In this study, we found that deficiency of HSPA12A in mice worsened cardiac dysfunction post-MI, and consistently, impaired post-MI angiogenesis was also detected in mouse hearts. The findings indicate that activating HSPA12A-dependent signaling might be beneficial for post-MI cardiac functional recovery by improving angiogenesis.

Angiogenesis could be either a physiological or a pathological process that forms new blood vessels trying to efficiently supply oxygen and nutrients during growth and development, as well as during wound healing after MI. Pathological angiogenesis is also essential for tumor progression and a variety of angiogenesis-related diseases (e.g., wet age-related macular degeneration, glaucoma, and diabetic retinopathy). Modulation of angiogenesis, therefore, is a promising therapeutic approach for related diseases. VEGF and Ang-1 have been regarded as crucial players in angiogenesis over several decades. Therapeutics targeting VEGF or Ang have been found to be clinically or preclinically useful for the suppression of pathological angiogenesis; however, two issues urgently need to be solved. First, there is still a lack of an ideal approach to promoting angiogenesis for improving post-MI wound healing; second, drug resistance is a current limitation of anti-VEGF therapy in clinical cancer treatment [6–11]. Therefore, identifying novel angiogenic factors remains a challenge for medical research. In this study, we found that HSPA12A, an atypical and distant member of the HSP70 family, showed upregulation during endothelial tube formation. Intriguingly, overexpression of HSPA12A promoted proliferation, migration, and tube formation of endothelial cells. By contrast, deficiency of HSPA12A impaired angiogenesis post-MI in mice. In supporting these results, HSPA12A displayed a positive regulation in angiogenic gene expression (VEGF, VEGFR2, and Ang-1). Altogether, our findings identified HSPA12A as a novel angiogenic activator.

AP-1 is a transcription factor regulating a variety of cellular processes such as proliferation, differentiation, and apoptosis. Recently, the emerging role of AP-1 has been demonstrated in angiogenesis and vascular development [13]. AP-1 is a dimer consisting of Jun (c-Jun, JunB, and JunD), ATF, or Fos. The transcription activity of AP-1 can be regulated by phosphorylation. For example, AP-1 can be phosphorylated by MAPKs and Akt, and the phosphorylated AP-1 will translocate to nuclei to drive the expression of VEGF and the VEGF downstream genes for angiogenesis [13–15, 38]. Conversely, VEGF can stimulate ERK activation [44]. Indeed, we found that overexpression of HSPA12A increases phosphorylation levels and nuclear localization of AP-1 in endothelial cells. We also found that HSPA12A activated ERKs and p38, whereas inhibition of ERKs and p38 diminished the HSPA12A-induced AP-1 phosphorylation, VEGF and VEGFR2 expression, and migration and tube formation of endothelial cells. The findings suggest that HSPA12A promoted angiogenesis through activating AP-1 in the p38- and ERK-dependent manners.

5. Conclusion

In summary, this study demonstrated that HSPA12A promotes in vitro angiogenesis and is required for myocardial angiogenesis post-MI in mice. This action was dependent, at least in part, on p38/ERK-mediated AP-1 signaling activation (Figure 12). The data suggest that HSPA12A expression may provide an effective strategy for post-MI cardiac functional recovery through promoting myocardial angiogenesis.

Data Availability

The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Authors’ Contributions

Tingting Li and Jun Wu contributed equally to this study.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (82170295, 82170851, 82000296, 81970692, 81870234, and 81770854) and by a project funded by the Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University.

Supplementary Materials

Supplementary 1. Figure S1: scheme of HSPA12A-adenovirus construction. Full length of mouse Hspa12a CDS was inserted in the multiple clonal sites (MCS).

Supplementary 2. Figure S2: overexpression of HSPA12A in HUVECs. Following infection with HSPA12A-adenovirus (Hspa12a^o/e) or empty adenovirus (NC) for 24 h, HSPA12A expression was evaluated by immunostaining for HSPA12A. DAPI was used to counterstain nuclei. . /group.

Supplementary 3. Figure S3: overexpression of HSPA12A in HPAEC promoted migration capacity. Following infection with HSPA12A-adenovirus (Hspa12a^o/e) or empty adenovirus (NC), a wound was made in the HPAEC monolayer. The healing of wounding was observed and expressed as the percentage of the original wounding area. vs. the time-matched NC. /group. .

Supplementary 4. Figure S4: effect of HSPA12A on c-Jun nuclear localization. Following infection with HSPA12A-adenovirus (Hspa12a^o/e) or empty adenovirus (NC) in HUVECs for 24 h, immunostaining for HSPA12A and c-Jun was performed. DAPI was used to counterstain nuclei. Yellow arrows indicate that the HUVECs with stronger HSPA12A staining also contained stronger c-Jun staining in nuclei, whereas white arrows indicate the opposite effects. . /group.

Supplementary 5. Figure S5: effect of HSPA12A on myocardial capillaries at basal levels. Cardiac tissues without infarction were collected at papillary muscle levels of mice. After cryosectioning was prepared, immunostaining for CD31 was performed to indicate capillaries. DAPI was used to counterstain nuclei. . /group. WT: wild-type mice; Hspa12a^-/-: HSPA12A knockout mice.

Supplementary 6. Figure S6: histological examination. Cardiac tissues were collected at papillary muscle levels after myocardial infarction for 14 days. After paraffin-embedded sectioning was prepared, hematoxylin-eosin (HE) staining was performed. . /group. WT: wild-type mice; Hspa12a^-/-: HSPA12A knockout mice.

References

L. Zhu, S. Lama, L. Tu, G. Dusting, J. Wang, and G. Liu, “TAK1 signaling is a potential therapeutic target for pathological angiogenesis,” Angiogenesis, vol. 24, no. 3, pp. 453–470, 2021.
View at: Publisher Site | Google Scholar
Y. Wang, J. Liu, Q. Kong et al., “Cardiomyocyte-specific deficiency of HSPB1 worsens cardiac dysfunction by activating NFκB-mediated leucocyte recruitment after myocardial infarction,” Cardiovascular Research, vol. 115, no. 1, pp. 154–167, 2019.
View at: Publisher Site | Google Scholar
J. Li, Y. Zhang, C. Li et al., “HSPA12B attenuates cardiac dysfunction and remodelling after myocardial infarction through an eNOS-dependent mechanism,” Cardiovascular Research, vol. 99, no. 4, pp. 674–684, 2013.
View at: Publisher Site | Google Scholar
W. Guo, W. Feng, J. Huang et al., “Supramolecular self-assembled nanofibers efficiently activate the precursor of hepatocyte growth factor for angiogenesis in myocardial infarction therapy,” ACS Applied Materials & Interfaces, vol. 13, no. 19, pp. 22131–22141, 2021.
View at: Publisher Site | Google Scholar
A. van der Laan, J. Piek, and N. van Royen, “Targeting angiogenesis to restore the microcirculation after reperfused MI,” Nature Reviews Cardiology, vol. 6, no. 8, pp. 515–523, 2009.
View at: Publisher Site | Google Scholar
R. Gianni-Barrera, A. Butschkau, A. Uccelli et al., “PDGF-BB regulates splitting angiogenesis in skeletal muscle by limiting VEGF-induced endothelial proliferation,” Angiogenesis, vol. 21, no. 4, pp. 883–900, 2018.
View at: Publisher Site | Google Scholar
E. Fagiani and G. Christofori, “Angiopoietins in angiogenesis,” Cancer Letters, vol. 328, no. 1, pp. 18–26, 2013.
View at: Publisher Site | Google Scholar
P. Saharinen, L. Eklund, and K. Alitalo, “Therapeutic targeting of the angiopoietin-TIE pathway,” Nature Reviews Drug Discovery, vol. 16, no. 9, pp. 635–661, 2017.
View at: Publisher Site | Google Scholar
S. Yoodee, P. Peerapen, S. Plumworasawat, and V. Thongboonkerd, “_ARID1A_ knockdown in human endothelial cells directly induces angiogenesis by regulating angiopoietin-2 secretion and endothelial cell activity,” International Journal of Biological Macromolecules, vol. 180, pp. 1–13, 2021.
View at: Publisher Site | Google Scholar
J. Wagner, C. Kline, L. Zhou, V. Khazak, and W. El-Deiry, “Anti-tumor effects of ONC201 in combination with VEGF-inhibitors significantly impacts colorectal cancer growth and survival in vivo through complementary non-overlapping mechanisms,” Journal of Experimental & Clinical Cancer Research, vol. 37, no. 1, p. 11, 2018.
View at: Publisher Site | Google Scholar
L. Lestable, P. Gabrielle, A. Bron, P. Nguyen, and C. Creuzot-Garcher, “Resultats a 1 an d'un traitement par injections intra-vitreennes d'agent anti- vegf dans la degenerescence maculaire liee a l'age exsudative naive : donnees d'une etude du registre fight for retinal blindness,” Journal francais d’Ophtalmologie, vol. 43, no. 8, pp. 761–769, 2020.
View at: Publisher Site | Google Scholar
V. Anttila, A. Saraste, J. Knuuti et al., “Synthetic mRNA encoding VEGF-A in patients undergoing coronary artery bypass grafting: design of a phase 2a clinical trial,” Molecular therapy Methods & clinical development, vol. 18, pp. 464–472, 2020.
View at: Publisher Site | Google Scholar
Y. Yoshitomi, T. Ikeda, H. Saito-Takatsuji, and H. Yonekura, “Emerging role of AP-1 transcription factor JunB in angiogenesis and vascular development,” International journal of molecular sciences, vol. 22, no. 6, p. 2804, 2021.
View at: Publisher Site | Google Scholar
W. Dong, Y. Li, M. Gao et al., “IKKα contributes to UVB-induced VEGF expression by regulating AP-1 transactivation,” Nucleic Acids Research, vol. 40, no. 7, pp. 2940–2955, 2012.
View at: Publisher Site | Google Scholar
H. Menden, S. Welak, S. Cossette, R. Ramchandran, and V. Sampath, “Lipopolysaccharide (LPS)-mediated angiopoietin-2-dependent autocrine angiogenesis is regulated by NADPH oxidase 2 (Nox2) in human pulmonary microvascular endothelial cells,” The Journal of Biological Chemistry, vol. 290, no. 9, pp. 5449–5461, 2015.
View at: Publisher Site | Google Scholar
Z. Han, Q. A. Truong, S. Park, and J. L. Breslow, “Two Hsp 70 family members expressed in atherosclerotic lesions,” Proceedings of the National Academy of Sciences of the United States of America, vol. 100, no. 3, pp. 1256–1261, 2003.
View at: Publisher Site | Google Scholar
J. L. Pongrac, F. A. Middleton, L. Peng, D. A. Lewis, P. Levitt, and K. Mirnics, “Heat shock protein 12A shows reduced expression in the prefrontal cortex of subjects with schizophrenia,” Biological Psychiatry, vol. 56, no. 12, pp. 943–950, 2004.
View at: Publisher Site | Google Scholar
Y. Mao, Q. Kong, R. Li et al., “Heat shock protein A12A encodes a novel prosurvival pathway during ischaemic stroke,” Biochimica et Biophysica Acta - Molecular Basis of Disease, vol. 1864, no. 5, pp. 1862–1872, 2018.
View at: Publisher Site | Google Scholar
X. Zhang, X. Chen, T. Qi et al., “HSPA12A is required for adipocyte differentiation and diet-induced obesity through a positive feedback regulation with PPARγ,” Cell Death and Differentiation, vol. 26, no. 11, pp. 2253–2267, 2019.
View at: Publisher Site | Google Scholar
Q. Kong, N. Li, H. Cheng et al., “HSPA12A is a novel player in nonalcoholic steatohepatitis via promoting nuclear PKM2-mediated M1 macrophage polarization,” Diabetes, vol. 68, no. 2, pp. 361–376, 2019.
View at: Publisher Site | Google Scholar
H. Cheng, X. Cao, X. Min et al., “Heat-shock protein A12A is a novel PCNA-binding protein and promotes hepatocellular carcinoma growth,” The FEBS Journal, vol. 287, no. 24, pp. 5464–5477, 2020.
View at: Publisher Site | Google Scholar
Y. Dai, J. Liu, X. Zhang et al., “HSPA12A improves endothelial integrity to attenuate lung injury during endotoxemia through activating ERKs and Akt-dependent signaling,” International Immunopharmacology, vol. 99, p. 107987, 2021.
View at: Publisher Site | Google Scholar
J. Liu, S. Du, Q. Kong et al., “HSPA12A attenuates lipopolysaccharide-induced liver injury through inhibiting caspase-11-mediated hepatocyte pyroptosis via PGC-1α-dependent acyloxyacyl hydrolase expression,” Cell Death and Differentiation, vol. 27, no. 9, pp. 2651–2667, 2020.
View at: Publisher Site | Google Scholar
S. Lin, Y. Wang, X. Zhang et al., “HSP27 alleviates cardiac aging in mice via a mechanism involving antioxidation and mitophagy activation,” Oxidative Medicine and Cellular Longevity, vol. 2016, Article ID 2586706, 13 pages, 2016.
View at: Publisher Site | Google Scholar
J. Wu, X. Li, L. Huang et al., “HSPA12B inhibits lipopolysaccharide-induced inflammatory response in human umbilical vein endothelial cells,” Journal of Cellular and Molecular Medicine, vol. 19, no. 3, pp. 544–554, 2015.
View at: Publisher Site | Google Scholar
M. He, W. Shi, M. Yu et al., “Nicorandil attenuates LPS-induced acute lung injury by pulmonary endothelial cell protection via NF-κB and MAPK pathways,” Oxidative Medicine and Cellular Longevity, vol. 2019, Article ID 4957646, 13 pages, 2019.
View at: Publisher Site | Google Scholar
H. Pulkkinen, M. Kiema, J. Lappalainen et al., “BMP6/TAZ-Hippo signaling modulates angiogenesis and endothelial cell response to VEGF,” Angiogenesis, vol. 24, no. 1, pp. 129–144, 2021.
View at: Publisher Site | Google Scholar
X. Min, X. Zhang, Y. Li et al., “HSPA12A unstabilizes CD147 to inhibit lactate export and migration in human renal cell carcinoma,” Theranostics, vol. 10, no. 19, pp. 8573–8590, 2020.
View at: Publisher Site | Google Scholar
Q. Kong, L. Dai, Y. Wang et al., “HSPA12B attenuated acute myocardial ischemia/reperfusion injury via maintaining endothelial integrity in a PI3K/Akt/mTOR-dependent mechanism,” Scientific Reports, vol. 6, no. 1, p. 33636, 2016.
View at: Publisher Site | Google Scholar
L. Xu, R. Willumeit-Römer, and B. Luthringer-Feyerabend, “Effect of magnesium-degradation products and hypoxia on the angiogenesis of human umbilical vein endothelial cells,” Acta Biomaterialia, vol. 98, pp. 269–283, 2019.
View at: Publisher Site | Google Scholar
C. Huang, H. Kuo, P. Wu et al., “Soluble delta-like 1 homolog (DLK1) stimulates angiogenesis through Notch 1/Akt/eNOS signaling in endothelial cells,” Angiogenesis, vol. 21, no. 2, pp. 299–312, 2018.
View at: Publisher Site | Google Scholar
L. Kempers, Y. Wakayama, I. van der Bijl et al., “The endosomal RIN2/Rab5C machinery prevents VEGFR2 degradation to control gene expression and tip cell identity during angiogenesis,” Angiogenesis, vol. 24, no. 3, pp. 695–714, 2021.
View at: Publisher Site | Google Scholar
N. A. Abdel-Malak, C. B. Srikant, A. S. Kristof, S. A. Magder, J. A. Di Battista, and S. N. Hussain, “Angiopoietin-1 promotes endothelial cell proliferation and migration through AP-1-dependent autocrine production of interleukin-8,” Blood, vol. 111, no. 8, pp. 4145–4154, 2008.
View at: Publisher Site | Google Scholar
M. Saito, M. Hamasaki, and M. Shibuya, “Induction of tube formation by angiopoietin-1 in endothelial cell/fibroblast co-culture is dependent on endogenous VEGF,” Cancer Science, vol. 94, no. 9, pp. 782–790, 2003.
View at: Publisher Site | Google Scholar
Y. S. Park, G. Kim, Y. M. Jin, J. Y. Lee, J. W. Shin, and I. Jo, “Expression of angiopoietin-1 in hypoxic pericytes: regulation by hypoxia- inducible factor-2α and participation in endothelial cell migration and tube formation,” Biochemical and Biophysical Research Communications, vol. 469, no. 2, pp. 263–269, 2016.
View at: Publisher Site | Google Scholar
C. Fu, R. Tyagi, A. Chin et al., “Inositol polyphosphate multikinase inhibits angiogenesis via inositol pentakisphosphate-induced HIF-1α degradation,” Circulation Research, vol. 122, no. 3, pp. 457–472, 2018.
View at: Publisher Site | Google Scholar
C. Lee, S. Chen, S. Tsai et al., “Hyperbaric oxygen induces VEGF expression through ERK, JNK and c-Jun/AP-1 activation in human umbilical vein endothelial cells,” Journal of Biomedical Science, vol. 13, no. 1, pp. 143–156, 2006.
View at: Publisher Site | Google Scholar
F. Zhang, K. Li, M. Pan et al., “miR-589 promotes gastric cancer aggressiveness by a LIFR-PI3K/AKT-c-Jun regulatory feedback loop,” Journal of experimental & clinical cancer research: CR, vol. 37, no. 1, p. 152, 2018.
View at: Publisher Site | Google Scholar
L. Seclì, F. Fusella, L. Avalle, and M. Brancaccio, “The dark-side of the outside: how extracellular heat shock proteins promote cancer,” Cellular and Molecular Life Sciences: CMLS, vol. 78, no. 9, pp. 4069–4083, 2021.
View at: Publisher Site | Google Scholar
M. J. Vos, J. Hageman, S. Carra, and H. H. Kampinga, “Structural and functional diversities between members of the human HSPB, HSPH, HSPA, and DNAJ chaperone families,” Biochemistry, vol. 47, no. 27, pp. 7001–7011, 2008.
View at: Publisher Site | Google Scholar
A. Henriques, V. Koliaraki, and G. Kollias, “Mesenchymal MAPKAPK2/HSP27 drives intestinal carcinogenesis,” Proceedings of the National Academy of Sciences of the United States of America, vol. 115, no. 24, pp. E5546–E5555, 2018.
View at: Publisher Site | Google Scholar
K. Rashmi, H. Atreya, M. Harsha Raj, B. Salimath, and H. Aparna, “A pyrrole-based natural small molecule mitigates HSP90 expression in MDA-MB-231 cells and inhibits tumor angiogenesis in mice by inactivating HSF-1,” Cell Stress & Chaperones, vol. 22, no. 5, pp. 751–766, 2017.
View at: Publisher Site | Google Scholar
X. Wang, H. Gu, W. Huang et al., “Hsp20-mediated activation of exosome biogenesis in cardiomyocytes improves cardiac function and angiogenesis in diabetic mice,” Diabetes, vol. 65, no. 10, pp. 3111–3128, 2016.
View at: Publisher Site | Google Scholar
K. Watari, T. Shibata, H. Fujita et al., “NDRG1 activates VEGF-A-induced angiogenesis through PLCγ1/ERK signaling in mouse vascular endothelial cells,” Communications biology, vol. 3, no. 1, p. 107, 2020.
View at: Publisher Site | Google Scholar

Copyright

Copyright © 2022 Tingting Li et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

PDF Download Citation

Download other formats

Order printed copies

Views

275

Downloads

359

Citations