Engineered myocardium model to study the roles of HIF-1α and HIF1A-AS1 in paracrine-only signaling under pathological level oxidative stress

Abstract

Studying heart tissue is critical for understanding and developing treatments for cardiovascular diseases. In this work, we fabricated precisely controlled and biomimetic engineered model tissues to study how cell-cell and cell-matrix interactions influence myocardial cell survival upon exposure to pathological level oxidative stress. Specifically, the interactions of endothelial cells (ECs) and cardiomyocytes (CMs), and the role of hypoxia inducible factor-1α (HIF-1α), with its novel alternative regulator, HIF-1α antisense RNA1 (HIF1A-AS1), in these interactions were investigated. We encapsulated CMs in photo-crosslinkable, biomimetic hydrogels with or without ECs, then exposed to oxidative stress followed by normoxia. With precisely controlled microenvironment provided by the model tissues, cell-cell interactions were restricted to be solely through the secreted factors. CM survival after oxidative stress was significantly improved, in the presence of ECs, when cells were in the model tissues that were functionalized with cell attachment motifs. Importantly, the cardioprotective effect of ECs was reduced when HIF-1α expression was knocked down suggesting that HIF-1α is involved in cardioprotection from oxidative damage, provided through secreted factors conferred by the ECs. Using model tissues, we showed that cell survival increased with increased cell-cell communication and enhanced cell-matrix interactions. In addition, whole genome transcriptome analysis showed, for the first time to our knowledge, a possible role for HIF1A-AS1 in oxidative regulation of HIF-1α. We showed that although HIF1A-AS1 knockdown helps CM survival, its effect is overridden by CM-EC bidirectional interactions as we showed that the conditioned media taken from the CM-EC co-cultures improved CM survival, regardless of HIF1A-AS1 expression.

Statement of significance: Cardiovascular diseases, most of which are associated with oxidative stress, is the most common cause of death worldwide. Thus, understanding the molecular events as well as the role of intercellular communication under oxidative stress is upmost importance in its prevention. In this study we used 3D engineered tissue models to investigate the role of HIF-1α and its regulation in EC-mediated cardioprotection. We showed that EC-mediated protection is only possible when there is a bidirectional crosstalk between ECs and CMs even without physical cell-cell contact. In addition, this protective effect is at least partially related to cell-ECM interactions and HIF-1α, which is regulated by HIF1A-AS1 under oxidative stress.

Keywords: Cardiomyocyte; Endothelial cell; Hypoxia inducible factor; Oxidative stress; Tissue engineering.

Figure 1

Characterization of hiPSC-derived ECs (A-E). (A) The bright field images of hiPSCs at days 1 and 6 during differentiation (Scale bar: 100 μm). (B) qPCR analysis for EC marker genes in iECs and HUVECs (positive control). (C) Immunostaining images of iECs, HUVECs, and hiPSCs (negative control) for EC markers: CD31, vWF, and VE-Cadherin (top to down) (Scale bar: 200 μm). (D) qPCR analysis for pluripotency markers NANOG and OCT-4 in HUVECs, hiPSCs and iECs. (E) Bright field images of tube formation assay of iECs and HUVECs (Scale bar: 200 μm). (F) qPCR analysis of shRNA treated HUVECs and iECs showing the downregulation of Hif-1α at mRNA level. (G) ELISA results showing the relative HIF-1α protein concentration of HIF-1α shRNA treated HUVECs and iECs. (* indicates statistical significance between two individual groups (p<0.05) and ** indicates significant difference between a single group to all other groups (p<0.01), n≥3 for all).

Figure 2

Conventional culture of CMs with or without iECs under oxidative stress and normoxia. An improvement in cell survival was observed with the presence of ECs and this improvement was compromised with HIF-1α knock down. (A) The Live/Dead Assay (green: live, red: dead, blue: live iECs) images. (B) The live cell percentages of CM-alone cultures that received iEC-only or iEC-CM co-culture-conditioned media, or non-conditioned media, under normoxia or oxidative stress. (C) The cell survival of single cultures and co-cultures of CMs and HIF-1α shRNA knockdown or control iECs after oxidative stress treatment. (D) The live CM:EC ratio of the different co-cultures at the end of normoxia or oxidative stress treatment. (* indicates statistical significance between two individual groups (p<0.05), and ** indicates significant difference between a single group to all other groups (p<0.05), n≥3 for all) (Scale bar=50 μm).

Figure 3

Response of ECs and CMs to oxidative stress in 3D. 3D environment improved CM survival, however, there was a more significant increase in survival with iEC presence. This cardioprotective effect was compromised with HIF-1α knock down, consistent with the 2D results. (A) The schematic of 3D construct preparation. (B) The Live/Dead Assay images and (C) the cell survival of the single cultures and co-cultures of CMs and HIF-1α shRNA knockdown or control iECs after oxidative stress treatment. (* indicates statistical significance between two individual groups (p<0.05), n≥3 for all) (Scale bar=100 μm).

Figure 4

The effect of cell-ECM interactions on cell survival. The importance of 3D environment and the cell-cell distance is shown with improved survival in 3D cultures with high encapsulation density. (A) The comparison of cell survival in 2D vs. 3D culture conditions. (B) The comparison of cell survival of EC-only and CM-only cultures in 3D with different cell encapsulation densities. (C) The live/dead images of iEC-CM model tissues in PEG and PEG-RGD, under normoxia or oxidative stress (Ox. stress) (Insets show magnified images of the corresponding samples). (D) The cell survival (%) of CMs, iECs and iECCM co-cultures encapsulated in PEG vs. PEG-RGD hydrogels in Fn (-) media. (* indicates statistical significance between two individual groups (p<0.05), N.S. indicates “no significance” (p>0.05), n≥3 for all). (Scale bar=100 μm, inset scale bar=20 μm).

Figure 5

The effect of EC presence and the role of HIF-1α on apoptosis under oxidative stress. (A) TUNEL assay images of CM and iEC-CM model tissues under oxidative stress (Blue: DAPI; Green: fragmented DNA) (white triangles indicate TUNEL positive cells) (B) TUNEL positive cell percentages of CM and iEC-CM model tissues under normoxia and oxidative stress. (C) Caspase 3/7 activity of CM, iEC-CM, and HIF-1α shRNA knockdown iEC-CM model tissues under oxidative stress (* indicates statistical significance between two individual groups and ** indicates significant difference between a single group to all other groups (p<0.05), # indicates no statistical significance compared to “CM under normoxia” group (p<0.05), n≥3 for all).

Figure 6

Changes in expression of HIF-1α related factors with oxidative stress treatment and EC presence. The results confirm the bidirectional crosstalk between iECs and CMs as the most drastic changes were observed as a result of the co-culture conditions. (A) Relative HIF1A-AS1 mRNA expression in iEC-only or iEC-CM co-culture tissue constructs after normoxia (N) or oxidative stress treatment (S). (B) Normalized TGF-β3 protein concentrations in CM-alone tissue constructs before oxidative stress treatment (before), after 16h H₂O₂ treatment (16h) and after the subsequent 2h normoxia treatment (2h). (C) Normalized TGF-β3 protein concentrations in CM-alone cultures that received non-conditioned or iEC-only or iEC-CM co-culture-conditioned media, under normoxia (N) or oxidative stress (S). (* indicates statistical significance between two individual groups and ** indicates significant difference between a single group to all other groups (p<0.05), N.S. indicates “no significance” (p>0.05), n≥3 for all).

Figure 7

Effect of HIF1A-AS1 expression and CM-EC interactions on cell survival. (A) The time table for the experiment (“N” stands for normoxia and “S” stands for oxidative stress). The RT-qPCR analysis showing HIF1A-AS1 mRNA expression of control or HIF1A-AS1 siRNA transfected iECs (B) right after transfection, and (C) 16h after transfection. (D) Live cell (%) of single CM cultures incubated for 16h in iECs only or CM-iEC co-culture pre-conditioned media prepared under normoxia (N) or oxidative stress (S). (* indicates statistical significance between two individual groups and ** indicates significant difference between a single group to all other groups (p<0.05), n≥3 for all).