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. 2019 Sep 19;18(21):2998–3009. doi: 10.1080/15384101.2019.1666611

HIF1α/miR-199a/ADM feedback loop modulates the proliferation of human dermal microvascular endothelial cells (HDMECs) under hypoxic condition

Yang Sun 1, Xiang Xiong 1,*, Xiancheng Wang 1,✉
PMCID: PMC6791693  PMID: 31537150

ABSTRACT

Hypoxia-inducible factor 1α (HIF1α) plays a protective role in the hypoxia-induced cellular injury. In the present study, we attempted to investigate the role and mechanism of HIF1αin human dermal microvascular endothelial cells (hDMECs), a common-used cell model for researches on the hypoxia-induced injury during skin wounds healing. As revealed by ChIP and online tools prediction and confirmed by luciferase reporter and ChIP assays, HIF1A can bind to the promoter regions of ADM and miR-199a, while miR-199a directly binds to the 3ʹUTR of HIF1A and ADM. Hypoxia stress induces HIF1α and ADM expression while inhibits miR-199a expression. Under hypoxic condition, HIF1α knockdown increases the nucleus translocation of p65 and the release of TNF-α and IL-8, inhibits the proliferation and migration, while promotes the cellular permeability in HDMECs upon hypoxic stress, while ADM overexpression and miR-199a inhibition exerted an opposite effect on HDMECs. ADM overexpression or miR-199a inhibition could partially reverse the effect of HIF1A knockdown under hypoxia. In summary, we demonstrate a feedback loop consists of HIF1α, miR-199a, and ADM which protect HDMECs from hypoxia-induced cellular injury by modulating the inflammation response, cell proliferation, migration and permeability in HDMECs.

KEYWORDS: Hypoxia-inducible factor 1α (HIF1A), human dermal microvascular endothelial cells (HDMECs), adrenomedullin (ADM), miR-199a, Hypoxia

Introduction

The healing of human skin wounds is a complicated and highly coordinated biological process [1]. After an injury occurs due to accidental trauma, empyrosis, surgical treatment or on the basis of diseases like diabetic foot ulcerations, the following healing sequence depends on different stimuli, which might be disturbed by multi-factors.

Hypoxia is a characteristic condition during wound healing which is immediately established after a skin injury, due to blood vessel damage or collapse. In situations of low oxygen tension, not only will there be more necrotic debris to facilitate bacterial growth but the primary mechanism of the immune system in combating these microbes is compromised [2,3]. Hypoxia might disturb the cellular interplay of endothelial cells and subcutaneous fibroblasts, which both are the most important cell types involved in wound healing and necessary for the re-establishment of the tissue homeostasis after repair [4,5].

However, an environment with low oxygen tension has been reported to activate factors such as hypoxia-inducible factor-1 (HIF1) [6,7] and also increases the rate of re-epithelialization [8]. Restoration of cellular homeostasis in the hypoxic environment is orchestrated by the transcription factor HIF-1 [9]. Of the most intensively studied HIFA genes, HIF1α has a ubiquitous pattern of expression in all tissues [10], whereas HIF2α expression is restricted to specific cell types [11,12]. Recently, using mice with a targeted HIF1A deficiency in keratinocytes, Rezvani et al. revealed that HIF1α plays a vital role in skin homeostasis during aging and that loss of HIF1α leads to a delay in wound healing [13]. Increasing expression of HIF1α could shorten the time of wound closure in diabetic wound healing mice model [14]. As a vasoactive peptide, adrenomedullin (ADM) could be increased by hypoxia and is a canonical HIF1-responsive gene [15]. Moreover, ADM plays critical roles in regulations of angiogenesis, vascular stability and permeability, which are essential for wound healing [16,17]. Although HIF1α may serve as a critical protector in wound healing, its specific effect on human dermal microvascular endothelial cells (HDMECs), which are commonly used to simulate the hypoxia-induced injury during wound healing [18,19], and the underlying mechanism is yet unclear.

In addition to HIF1α, the role of microRNAs (miRNAs) in hypoxia-induced cellular injury has drawn much attention. MiR-199a, a widely-reported tumor suppressor [20,21], has been reported to be acutely downregulated in cardiac myocytes on a decline in oxygen tension, and this reduction is required for the rapid upregulation of its target, HIF1α [22]. The above findings inspire us to speculate that miR-199a may play a role in HDMECs upon hypoxia-induced injury via regulating HIF1α.

To investigate the specific effect and mechanism of HIF1α in hypoxia-induced cellular injury in HDMECs, we analyzed the downstream genes of HIF1A by chromatin immunoprecipitation (ChIP) (Table S1) and found that its downstream target gene adrenomedullin (ADM) can promote the proliferation and migration of endothelial cells, and regulate vascular permeability [23,24]. Moreover, online tools predicted that HIF1α could target the promoter region of miR-199a, while miR-199a can target HIF1A and ADM 3ʹUTR. Therefore, we speculate that the above factors can constitute a regulatory network to modulate the proliferation, permeability of HDMECs under hypoxic condition, thereby protecting the vitality of HDMECs during hypoxia-induced injury.

In the present study, the expression patterns of HIF1α and ADM were first determined under hypoxia condition. Next, the dynamic effect of HIF1α and ADM on inflammation response, cell proliferation, migration and permeability in HDMECs were evaluated under hypoxia stress. After that, the predicted bindings of HIF1A, ADM, and miR-199a were validated. Finally, the dynamic effect of HIF1α and miR-199a on HDMECs under hypoxia condition was examined. In summary, we attempted to provide a novel mechanism by which the HIF1α/miR-199a/ADM feedback loop protects HDMECs from hypoxia-induced injury.

Materials and methods

Cell line, cell culture, and hypoxia treatment

HDMECs were obtained from Sciencell (San Diego, CA, USA) and cultured in an endothelial cell medium (Sciencell) containing 5% FBS, 1% endothelial cell growth supplement, and 1% penicillin/streptomycin solution at a density of 105 cells/ml and placed in a humidified CO2 incubator at 37°C until cells reached 70%-80% confluence. For the construction of hypoxia-induced injury model, HDMECs were incubated in an airtight chamber with a gas mixture of 5% CO2, 85–94.5% N2 and 0.5-10% O2. HDMECs exposed to 0.5%, 1%, 3% and 10%, O2 for 12 h or exposed to 1% O2 for 0, 6, 12, and 24 h.

HIF1A knockdown was achieved by transfection of si-HIF1A (final concentration is 50 nM) with siRNA-Mate transfection reagent (GeneCopoecia, Guangzhou, China). HIF1A and ADM overexpression were achieved by transfection of HIF1A and ADM – pCDNA3.1 expression vectors via lipofectamine 2000 (Invitrogen). The expression vectors were constructed by PCR. The primers were listed in Table S2. The expression of miR-199a was achieved by transfection of miR-199a mimics or inhibitor at a final concentration of 50 nM (Genepharma, Shanghai, China) with the help of Lipofectamine 2000 (Invitrogen).

Real-time PCR

Total RNA was extracted using Trizol reagent (Invitrogen) following manufacturer’s instruction. By using miRNA-specific Stem-Loop RT primer, total RNA was reverse transcribed, and the miScript Reverse Transcription kit (Qiagen, Germany) was used for miRNA qRT-PCR. The RNU6B expression was used as an endogenous control. The SYBR green PCR Master Mix (Qiagen) was used for mRNA and miRNA expression detection following the protocol. The β-actin expression was used as an endogenous control. The 2−ΔΔCT method was used to analyze the relative fold changes. (Primers were shown in Table S2)

Immunoblotting

The protein levels of HIF1α, ADM, and p65 were detected by immunoblotting following the method described previously [25] with the following antibodies at 4°C overnight: anti-HIF1α (dilution 1:1000, ab82832, Abcam, Cambridge, MA, USA), anti-ADM (dilution 1:1000, ab69117, Abcam), and anti-p65 (dilution 1:1000, ab16502, Abcam) and incubated with the HRP-conjugated secondary antibody (1:5000, Santa Cruz, USA). Signals were visualized using ECL Substrates (Millipore, USA). The protein density was measured by ImageJ software (NIH, USA) and expression was normalized to endogenous β-actin (n = 3).

Luciferase reporter assay

For determination of miR-199a interacting with HIF1A and ADM, the fragment of HIF1A and ADM 3ʹUTR was amplified by PCR and cloned to the downstream of the Renilla psiCHECK2 vector (Promega, Madison, WI, USA), named wt-HIF1A 3ʹUTR and wt-ADM 3ʹUTR. To generate the HIF1A and ADM mutant reporter, the seed region of the HIF1A and ADM was mutated to remove the complementarity to miR-199a, named mut-HIF1A 3ʹUTR and mut-ADM 3ʹUTR. HEK293 cells (ATCC, USA) were co-transfected with the indicated vectors and miR-199a mimics or miR-199a inhibitor. Luciferase assays were performed 48 h after transfection using the Dual-Luciferase Reporter Assay System (Promega). Renilla luciferase activity was normalized to Firefly luciferase activity for each transfected well.

For determination of transcriptional activity, the miR-199a promoter fragments containing HIF1α binding sites were PCR cloned into Renilla psiCHECK2 vector. The mutant fragments were generated by recombinant PCR (mutated 6 bases). The primers were listed in Table S2. HDMECs were seeded at 5 × 105 cells/dish and incubated overnight at 37°C in a 5% CO2 incubator. For each transfection, empty or expression vectors (4, 8, 16 µg) along with the promoter-luciferase DNA (0.3 mg) were mixed in Opti-MEM (0.2 ml) and a precipitate was formed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Cells were washed with Opti-MEM, and complexes were applied to the cells. After transfection for 24 h, cells were harvested, and extracts were prepared with the Glo Lysis Buffer (Promega, Madison, WI, USA). Luciferase activity was measured in extracts from triplicate samples using the Bright-Glo Luciferase Assay system (Promega).

DNA synthesis determination by EdU assay

DNA synthesis was determined according to the incorporation of the thymidine analog 5-ethynyl 2-deoxyuridine (EdU) into genomic DNA by using the Click-IT EdU Alexa Fluor 488 kit for flow cytometry (Invitrogen). Cells were treated or transfected, and growth medium was replaced with medium containing EdU (10 μmol/L). Two hours later, the medium was replaced with EdU–free medium. Then an Apollo staining and DAPI staining were performed to detect the EdU positive cells with a fluorescence microscope following the protocols. The EdU incorporation rate calculated as the ratio of EdU-positive to total DAPI-positive cells (blue cells).

Permeability assays

HDMECs were seeded in 4 mm pore size and 12 mm diameter transwells at 1 × 105 cells/well for 3 days. The confluent monolayers were transfected with si-HIF1A, ADM expression vector or miR-199a inhibitor for 24 h. 0.3 mg/ml Evans blue (Sigma-Aldrich) containing 2% BSA (Sigma-Aldrich) were added to the upper chamber. Fresh growth medium was added to the lower chamber. After 10 min, the OD650 was measured in the lower chamber.

Wound healing assays

Transfected HDMECs were seeded into 24-well plates and allowed to grow to 60% confluent in the complete medium. Cells were then wounded by a sterile pipette tip, washed with PBS to remove cell debris. The healing process was observed by microscopy at 24 h. The migration distance = the wound width of 0 h- the wound width 24 h.

Chromatin immunoprecipitation (ChIP)

Treated or transfected cells were cross-linked with 1% formaldehyde, sheared to an average size of 400 bp DNA, and immunoprecipitated using antibodies against HIF1α. The ChIP-PCR primers were designed to amplify the miR-199a promoter regions containing putative HIF1α binding sites. A positive control antibody (RNA polymerase II) and negative control (NC) nonimmune IgG were used to demonstrate the efficacy of the kit reagents (Epigentek Group Inc., NY, USA, P-2025-48). The immunoprecipitated DNA was subsequently cleaned, released, and eluted. The eluted DNA was used for ChIP-PCR. The fold-enrichment (FE) was calculated as the ratio of the amplification efficiency of the ChIP sample to that of the nonimmune IgG. The amplification efficiency of RNA Polymerase II was used as a positive control. FE% = 2 (IgG CT-Sample CT) × 100%.

Determination of cytokines by ELISA

HDMECs culture medium was collected for ELISA assay using human TNF-αand IL-8 ELISA kits according to the manufacturer’s instructions (Santa Cruz Biotechnology, Santa Cruz, CA, USA). 100 μl serially-diluted standard samples or culture medium supernatant samples were added into the specific antibody coated-microplate and were incubated at 37°C for 1 h. The plate was updated with 100 μl horseradish peroxidase-linked secondary antibody for incubation at 37°C for 30 min and washed with 100 μl PBS with Tween 20 (PBST) three times before incubation. Finally, the plate was inoculated with 100 μl substrate at dark for 15 min; the specific binding optical density was assayed immediately at 450 nm with a spectrophotometer (Bio-Rad Laboratories).

Statistical analysis

Data were expressed as means ± SD of at least three independent experiments and statistically analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test or independent sample t-test using the SPSS Statistics 17.0 software. The level of significance was based on the probability of P < 0.05, P < 0.01.

Results

HIF1α induces ADM expression in HDMECs under hypoxia

To confirm the expression pattern of HIF1α and ADM under hypoxia condition, HDMECs were exposed to 0.5%, 1%, 3%, 10%, and 21% O2 for 12 h or exposed to 1% O2 for 0, 6, 12, and 24 h, and examined for the protein levels of HIF1A and ADM. Figure 1(a,b) shows that hypoxia stress can induce protein levels of HIF1A and ADM in a time- and concentration-dependent manner.

Figure 1.

Figure 1.

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HIF1α induces ADM expression in HDMECs under hypoxia. HDMECs were exposed to 0.5%, 1%, 3%, 10%, and 21% O2 for 12 h or exposed to 1% O2 for 0, 6, 12, and 24 h, and examined for HIF1α and ADM expression by Immunoblotting (a and b), HIF1A expression achieved by transfection of si-HIF1A and HIF1A overexpressing vector, as confirmed by qPCR (c) and Immunoblotting (d) under 1% O2 stimulation. ADM mRNA expression (e) and protein levels (f) in response to HIF1A knockdown or overexpression determined using qPCR and Immunoblotting. (g) HIF1A mRNA expression in HDMECs transfected with 4, 8, and 16 µg HIF1A overexpressing vector determined by qPCR. (h) ADM mRNA expression these transfected HDMECs determined by qPCR. (i) The protein levels of HIF1α and ADM in these transfected HDMECs determined by Immunoblotting. *P < 0.05, **P < 0.01.

To examine the effect of HIF1α on ADM under hypoxia condition, we achieved HIF1α expression under hypoxia by transfection of si-HIF1A or HIF1A overexpressing vector, as confirmed by Immunoblotting (Figure 1(c-e)). In response to HIF1A knockdown, ADM mRNA expression and protein levels were significantly reduced (Figure 1(e,f)), on the contrary, HIF1A overexpression remarkably increased ADM mRNA expression and protein levels under hypoxia (Figure 1(e,f)). These data indicate that HIF1α positively regulates ADM expression under hypoxia.

Next, HDMECs were transfected with HIF1A overexpressing vector of different dose (4, 8, and 16 µg) and examined for transfection efficiency (Figure 1(h)). The mRNA expression and protein levels of ADM in these transfected HDMECs were detected. Figure 1(h,i) show that HIF1A overexpression can upregulate ADM mRNA and protein levels in a dose-dependent manner.

HIF1α regulates the cytokine release, cell proliferation, migration and permeability in HDMECs by modulating ADM transcriptional activity

Since online database JASPER predicted that HIF1α could bind to the promoter region of ADM, here, we performed the luciferase reporter assay to confirm the putative binding. As shown in Figure 2(a), the transcriptional activity of ADM was increased by the transfection of HIF1A overexpressing vector in a dose-dependent manner, more increased by 16 µg HIF1A. The data indicate that HIF1α binds to ADM promoter to activate its transcription. As a further confirmation, the mRNA expression and protein levels of ADM were detected in HDMECs co-transfected with si-HIF1A and ADM overexpressing vector under hypoxia condition. Consistently, ADM mRNA and protein level were remarkably reduced by HIF1A knockdown while increased by HIF1A overexpression, the effect of HIF1A knockdown on ADM could be partially attenuated by ADM overexpression (Figure 2(b,c)).

Figure 2.

Figure 2.

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HIF1α regulates the cytokine release, proliferation, permeability and migration in HDMECs by modulating ADM transcriptional activity. (a) HDMECs were co-transfected with pGL2-ADM promoter-LUC vector and 4, 8, and 16 µg HIF1A overexpressing vector and examined for promoter activity. HDMECs were co-transfected with si-HIF1A and ADM overexpressing vector under 1% O2 and examined for ADM mRNA expression by qPCR and protein levels by Immunoblotting (b-c), cytoplasm and nuclear p65 protein levels by Immunoblotting (d), the release of TNF-α and IL-8 by ELISA (e-f), cell proliferation by EdU (g), permeability by EB staining (g) and migration ability by wound healing assays (i). *P < 0.05, **P < 0.01, compared to control group; #P < 0.05, ##P < 0.01, compared to si-HIF1A + NC group.

ADM pretreatment can markedly reduce the nuclear translocation of p65, therefore decreasing the significantly increased gene expression levels and concentrations of cytokines [26]. Here, the dynamic effects of HIF1A knockdown and ADM overexpression on p65 nuclear translocation and cytokine release were evaluated under hypoxia. Consistent with the previous report, HIF1A knockdown increased, while ADM overexpression inhibited the nuclear translocation of p65 and the release of cytokines, including TNF-α and IL-8, under hypoxia (Figure 2(d,f)).

Next, cell proliferation, penetration migration were examined under the same circumstances. Under hypoxia, HIF1A knockdown inhibited, while ADM overexpression promoted HDMEC proliferation (Figure 2(g)). The penetration of HDMEC was significantly increased by HIF1A knockdown while suppressed by ADM overexpression (Figure 2(h)). The cell migration ability was also inhibited by HIF1A knockdown (Figure 2(i)). The effect of HIF1A knockdown could be partially attenuated by ADM overexpression (Figure 2(g-i)). These data indicate that HIF1α modulates the inflammation response, penetration and migration ability of HDMECs via regulating the transcriptional activity of ADM.

MiR-199a participates in HIF1A-mediated ADM regulation

miRNAs play a considerable role in the post-transcriptional regulation of gene expression [27]. In the present study, online tools (TargetScan and LncTar) predicted that HIF1A and ADM might be direct downstream targets of miR-199a (Figure 3(b,c)). Moreover, the online database JASPER predicted that the promoter region of miR-199a possesses two possible HIF1A binding sites (Figure 3(g)). These predictions suggest that HIF1α, miR-199a, and ADM may form a regulatory loop to participate in the inflammation response and penetration in HDMECs.

Figure 3.

Figure 3.

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miR-199a participates in HIF1α-mediated ADM regulation. (a) miR-199a expression in HDMECs achieved by transfection with miR-199a mimics or miR-199a inhibitor, as confirmed by qPCR. (b-c) The binding of miR-199a to HIF1A and ADM 3ʹ UTR confirmed by luciferase reporter assay. HIF1A and ADM mRNA expression in response to miR-199a inhibition or overexpression determined protein levels determined by Immunoblotting (d). (e) miR-199a expression under 21% and 1% O2 determined by qPCR. (f) Two putative HIF1αbinding sites within miR-199a promoter. Mutant-type luciferase reporter vector contained a 6 bp mutation in any or both of the two binding sites. (g) HEK293 cells were co-transfected with these vectors and HIF1A overexpressing vector and examined for luciferase activity. (h) ChIP assay performed with anti-HIF1A or anti-IgG to confirm the binding of HIF1α to miR-199a promoter. (i) miR-199a expression in HDMECs in response to HIF1A knockdown or HIF1A overexpression determined by qPCR. *P < 0.05, **P < 0.01, compared to control group; #P < 0.05, ##P < 0.01, compared to NC (negative control) group.

To validate the predicted bindings, first, we determined the transfection effective of miR-199a mimics or miR-199a inhibitor transfection using qPCR (Figure 3(a)). Then, the luciferase activities of wild- (wt-HIF1A 3ʹUTR and wt-ADM 3ʹUTR) and mutant-type (mut-HIF1A 3ʹUTR and mut-ADM 3ʹUTR) were determined in HEK293 cells. The luciferase activity of wild-type vectors’ could be significantly suppressed by miR-199a overexpression while enhanced by miR-199a inhibition, after mutating the predicted miR-199a binding sites, the alterations of the luciferase activity were abolished (Figure 3(b,c)). Moreover, miR-199a decreased the expression of HIF1α and ADM (Figure 3(d)). These findings suggested that HIF1A 3ʹUTR can serve as a sponge for miR-199a to counteract miR-199a-mediated repression of ADM.

Opposite to HIF1α and ADM, miR-199a expression was significantly reduced by hypoxia (Figure 3(e)), suggesting that HIF1α may suppress the transcriptional activity of miR-199a via binding to the putative binding sites within miR-199a promoter. Wild- and mutant-type miR-199a promoter reporter vectors were constructed. Mutant-type vectors contained the mutation(s) in any or both of the predicted HIF1αbinding sites all also constructed (Figure 3(f)). These vectors were co-transfected into HEK293 cells with HIF1A or NC (negative control) vector, and the promoter activity was examined. As shown in Figure 3(h), HIF1A overexpression caused a decrease in promoter activity of wild-type miR-199a reporter vector, while the suppressive effect of HIF1A was partially attenuated after mutating one of the predicted binding sites within miR-199a promoter, more attenuated when both sites mutated (Figure 3(g)). Furthermore, ChIP assay revealed that in precipitated HIF1α, the levels of miR-199a promoter site 1 and 2 were higher than in IgG immunoprecipitant, indicating that HIF1α binds to miR-199a promoter to block the transcription of miR-199a. Moreover, overexpression of HIF1A further rose the levels of miR-199a promoter site 1 and 2 in HIF1αimmunoprecipitant. As a further confirmation, qPCR revealed that HIF1A negatively regulated miR-199a expression (Figure 3(I)).

The dynamic effect of HIF1α/mir-199a/ADM feedback loop on HDMECs under hypoxia stress

After confirming the binding and regulation of HIF1α, miR-199a, and ADM, we co-transfected HDMECs with si-HIF1A, and miR-199a inhibitors and then examined the expression of ADM, cytoplasm p65, and nucleus p65, the release of TNF-α and IL-8, the proliferation and permeation of HDMECs under hypoxia. ADM mRNA expression and protein levels were significantly reduced by HIF1A knockdown while increased by miR-199a inhibition (Figure 4(a,b)). The nucleus translocation of p65 could be promoted by HIF1A knockdown while inhibited by miR-199a inhibition (Figure 4(c)). Consistently, the release of cytokines was increased by HIF1A knockdown while suppressed by miR-199a inhibition (Figure 4(d,e)). The above effects of miR-199a inhibition could all be partially reversed by HIF1A knockdown (Figure 4(a-e)).

Figure 4.

Figure 4.

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The dynamic effect of HIF1α/miR-199a/ADM feedback loop on HDMECs under hypoxia stress. HDMECs were co-transfected with si-HIF1A and miR-199a inhibitor under 1% O2 and examined for ADM mRNA expression by qPCR (a) and protein levels (b), cytoplasm and nuclear protein levels of p65 determined by Immunoblotting (c), the release of TNF-α and IL-8 determined by ELISA (d-e), cell proliferation determined by EdU (f), permeability determined by EB staining (g) and migration ability by wound healing assays (h). (i) A schematic diagram showing the feedback loop consists of HIF1α, miR-199a, and ADM protecting HDMECs against hypoxia-induced injury. *P < 0.05, **P < 0.01, compared to control group; #P < 0.05, ##P < 0.01, compared to si-HIF1A group.

Regarding the cellular functions, HIF1A knockdown significantly increased the HDMECs permeability while suppressed HDMEC proliferation and migration ability, oppositely, miR-199a inhibition reduced HDMECs permeability and migration ability while promoted proliferation (Figure 4(f-h)). Similarly, the effect of miR-199a could be partially reversed by HIF1A knockdown (Figure 4(f-h)). These data indicate that HIF1α, miR-199a, and ADM form a feedback loop, therefore protecting HDMECs from hypoxia stress via modulating the inflammation response, proliferation, migration and permeability in HDMECs (Figure 4(h)).

Discussion

In the present study, we demonstrate a feedback loop consists of HIF1α, miR-199a, and ADM which protect HDMECs from hypoxia-induced injury by modulating the inflammation response, cell proliferation, migration and permeability in HDMECs.

Based on the essential role of HIF1α in wound healing upon hypoxia-induced cellular injury [13,14,28], in the present study, we attempted to investigate the specific effect and mechanism of HIF1αin hypoxia-induced HDMEC injury. As shown by ChIP and luciferase assays, HIF1α directly binds to the promoter region of ADM to activate its transcription. ADM, an active peptide affirmed to be present extensively in vascular endothelium, heart, lung, and kidney [29], has a significant effect on the vascular protection, anti-inflammation, and intracellular signaling [30]. More importantly, ADM can be regulated by HIF1α and participates in HIF1α-mediated protection against hypoxia-induced injury [31,32]. Here, ADM mRNA expression and protein levels are positively regulated by HIF1α upon the hypoxic condition. Regarding the cellular functions, siRNA-mediated HIF1A knockdown causes a significant increase in the nucleus translocation of p65 and the subsequent increase in the release of TNF-α and IL-8, an inhibition on proliferation, and finally a promotion on cellular permeability in HDMECs upon hypoxic stress. On the contrary, ADM overexpression exerts an opposite effect on HDMECs and partially reversed the effect of HIF1A knockdown. These data indicate that HIF1α and ADM also cooperate with each other to protect HDMECs from hypoxia-induced injury.

In addition to protein-coding RNAs, the crucial role of miRNAs has been reported in hypoxia-induced injury [33]. MiRNAs regulate gene expression post-transcriptionally, mainly through interaction with 3´UTR of the target gene [34]. MiR-199a targeting HI1FA 3ʹUTR was wildly reported in several cancer types [35,36]. In hepatocellular carcinoma, the miR-199a level could be suppressed by overexpressed HIF1α under hypoxia [37]. In the present study, not only HIF1α but also ADM are direct downstream targets of miR-199a, which was described as a master regulator of a hypoxia-triggered pathway [22]. MiR-199a negatively regulates HIF1α and ADM via targeting the 3´UTR of them, indicating that HIF1α, miR-199a, and ADM may form a network to modulate the response of HDMECs to hypoxia.

Interestingly, miR-199a expression is inhibited by hypoxia stress, opposite to HIF1α and ADM. As also predicted by online tools, HIF1α as a transcriptional factor may bind to the promoter region of miR-199a, which was confirmed then by LUC and ChIP assays. Via binding to miR-199a promoter region, HIF1α also negatively regulates miR-199a expression under hypoxia. Consistent with their expression patterns, miR-199a inhibition exerts an opposite effect on HDMECs under hypoxia, which could be partially reversed by HIF1A knockdown, indicating that the feedback loop, consists of HIF1α, miR-199a, and ADM, can dynamically modulate the response of HDMECs to hypoxia-induced injury. In conclusion, we demonstrate a feedback loop HIF1α/miR-199a/ADM which protect HDMECs from hypoxia-induced cellular injury by modulating the inflammation response, cell proliferation, migration and permeability in HDMEC.

Funding Statement

This work was supported by the National Natural Science Foundation of China [No. 81671964];Natural Science Foundation of Hunan Province [2019JJ50847].

Acknowledgments

This study was supported by the National Natural Science Foundation of China (No. 81671964) and Natural Science Foundation of Hunan Province (2019JJ50847).

Author contribution statement

Yang Sun: performance of experiment, data analysis and manuscript writing work; Xiang Xiong: performance of experiment; Xiancheng Wang: study design and manuscript review. All authors read and approved the final manuscript version that was submitted for peer review.

Disclosure statement

No potential conflict of interest was reported by the authors.

Supplementary material

Supplemental data for this article can be accessed here.

Supplemental Material
Supplemental Material

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