Lactate in breast cancer cells is associated with evasion of hypoxia-induced cell cycle arrest and adverse patient outcome.

Tumor hypoxia is a common microenvironmental factor in breast cancers, resulting in stabilization of Hypoxia-Inducible Factor 1 (HIF-1), the master regulator of hypoxic response in cells. Metabolic adaptation by HIF-1 results in inhibition of citric acid cycle, causing accumulation of lactate in large concentrations in hypoxic cancers. Lactate can therefore serve as a secondary microenvironmental factor influencing cellular response to hypoxia. Presence of lactate can alter the hypoxic response of breast cancers in many ways, sometimes in opposite manners. Lactate stabilizes HIF-1 in oxidative condition, as well as destabilizes HIF-1 in hypoxia, increases cellular acidification, and mitigates HIF-1-driven inhibition of cellular respiration. We therefore tested the effect of lactate in MDA-MB-231 under hypoxia, finding that lactate can activate pathways associated with DNA replication, and cell cycling, as well as tissue morphogenesis associated with invasive processes. Using a bioengineered nano-patterned stromal invasion assay, we also confirmed that high lactate and induced HIF-1α gene overexpression can synergistically promote MDA-MB-231 dissemination and stromal trespass. Furthermore, using The Cancer Genome Atlas, we also surprisingly found that lactate in hypoxia promotes gene expression signatures prognosticating low survival in breast cancer patients. Our work documents that lactate accumulation contributes to increased heterogeneity in breast cancer gene expression promoting cancer growth and reducing patient survival.

Hypoxia-activated selectivity-improved anti-PKM2 antibody combined with prodrug TH-302 for potentiated targeting therapy in hepatocellular carcinoma.

Background: Hypoxia induces hepatocellular carcinoma (HCC) malignancies; yet it also offers treatment opportunities, exemplified by developing hypoxia-activated prodrugs (HAPs). Although HAP TH-302 combined with therapeutic antibody (Ab) has synergistic effects, the clinical benefits are limited by the on-target off-tumor toxicity of Ab. Here, we sought to develop a hypoxia-activated anti-M2 splice isoform of pyruvate kinase (PKM2) Ab combined with TH-302 for potentiated targeting therapy. Methods: Codon-optimized and hypoxia-activation strategies were used to develop H103 Ab-azo-PEG5k (HAP103) Ab. Hypoxia-activated HAP103 Ab was characterized, and hypoxia-dependent antitumor and immune activities were evaluated. Selective imaging and targeting therapy with HAP103 Ab were assessed in HCC-xenografted mouse models. Targeting selectivity, systemic toxicity, and synergistic therapeutic efficacy of HAP103 Ab with TH-302 were evaluated. Results: Human full-length H103 Ab was produced in a large-scale bioreactor. Azobenzene (azo)-linked PEG5k conjugation endowed HAP103 Ab with hypoxia-activated targeting features. Conditional HAP103 Ab effectively inhibited HCC cell growth, enhanced apoptosis, and induced antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) functions. Analysis of HCC-xenografted mouse models showed that HAP103 Ab selectively targeted hypoxic HCC tissues and induced potent tumor-inhibitory activity either alone or in combination with TH-302. Besides the synergistic effects, HAP103 Ab had negligible side effects when compared to parent H103 Ab. Conclusion: The hypoxia-activated anti-PKM2 Ab safely confers a strong inhibitory effect on HCC with improved selectivity. This provides a promising strategy to overcome the on-target off-tumor toxicity of Ab therapeutics; and highlights an advanced approach to precisely kill HCC in combination with HAP TH-302.

The Regulatory Mechanism of Hypoxia-inducible Factor 1 and its Clinical Significance.

Hypoxia-inducible factor (HIF) is a nuclear protein that plays a crucial role in oxygen homeostasis through its transcriptional activity and thousands of target gene profiles. Through transcriptional and post-transcriptional regulation, the downstream target genes of HIF can trigger multiple pathological responses in the body, including energy metabolism, cytopenia, and angiogenesis. There are three distinct subtypes of HIF: HIF-1, HIF-2, and HIF-3. HIF-1 is a significant regulator of the cellular response to hypoxia, and the balance between its production and degradation is critical for this response. As hypoxia is linked to several disorders, understanding HIF can open up novel avenues for the treatment of many diseases. This review describes the regulatory mechanisms of HIF-1 synthesis and degradation and the clinical significance of the hypoxia-inducible factor pathway in lung injury, kidney disease, hematologic disorders, and inflammation-related diseases.

Tissue-of-origin for cancers determines HIF-1 activation induced phenotypic heterogeneity.

Hypoxia-inducible factor-1 (HIF-1) is the master regulator of cellular response to hypoxia, and is activated in many cancers contributing to many steps in the metastatic cascade by acting as a key transcription co-regulator for a large number of downstream genes. Presence of hypoxia within a tumor is spatially nonuniform, and can also by dynamic. Further, although HIF-1 is primarily stabilized and activated by lack of molecular O2, its stability is also affected by other factors present in the tumor microenvironment. HIF-1 also crosstalks with other transcription factors in co-regulating gene expression. Consequently, it is nontrivial to predict the gene expression patterns in cells in response to hypoxia, or HIF-1 activation. Additionally, cancers originating from tissue origins with different basal level of partial oxygen tension may activate HIF-1 at different threshold of hypoxia. We analyzed large published single cell RNAseq data for colorectal, lung, and pancreatic cancers to investigate the phenotypic outcome of HIF-1 activation in cancer cells. We found that cancers from tissues with different partial O2 tension levels exhibit HIF-1 activation at different stages of metastasis, and phenotypically respond differently to HIF-1 activation, likely by contextual co-option of different transcription factors. We experimentally confirmed these predictions by using cell lines representative of colorectal, lung, and pancreatic cancers, finding that while hypoxia enhances growth of colorectal cancer, it induces increased invasion of lung, and pancreatic cancers. Our analysis suggest that HIF-1 activation may act as a rheostat regulating downstream gene expression towards phenotypic outcomes differently in various cancers.

The HIF-1α/EGF/EGFR Signaling Pathway Facilitates the Proliferation of Yak Alveolar Type II Epithelial Cells in Hypoxic Conditions.

The yak is a unique creature that thrives in low-oxygen environments, showcasing its adaptability to high-altitude settings with limited oxygen availability due to its unique respiratory system. However, the impact of hypoxia on alveolar type II (AT2) epithelial cell proliferation in yaks remains unexplored. In this study, we investigated the effects of different altitudes on 6-month-old yaks and found an increase in alveolar septa thickness and AT2 cell count in a high-altitude environment characterized by hypoxia. This was accompanied by elevated levels of hypoxia-inducible factor-1α (HIF-1α) and epidermal growth factor receptor (EGFR) expression. Additionally, we observed a significant rise in Ki67-positive cells and apoptotic lung epithelial cells among yaks inhabiting higher altitudes. Our in vitro experiments demonstrated that exposure to hypoxia activated HIF-1α, EGF, and EGFR expression leading to increased proliferation rates among yak AT2 cells. Under normal oxygen conditions, activation of HIF-1α enhanced EGF/EGFR expressions which subsequently stimulated AT2 cell proliferation. Furthermore, activation of EGFR expression under normoxic conditions further promoted AT2 cell proliferation while simultaneously suppressing apoptosis. Conversely, inhibition of EGFR expression under hypoxic conditions had contrasting effects. In summary, hypoxia triggers the proliferation of yak AT2 cells via activation facilitated by the HIF-1α/EGF/EGFR signaling cascade.

Human ESC-derived vascular cells promote vascular regeneration in a HIF-1α dependent manner.

Hypoxia-inducible factor (HIF-1α), a core transcription factor responding to changes in cellular oxygen levels, is closely associated with a wide range of physiological and pathological conditions. However, its differential impacts on vascular cell types and molecular programs modulating human vascular homeostasis and regeneration remain largely elusive. Here, we applied CRISPR/Cas9-mediated gene editing of human embryonic stem cells and directed differentiation to generate HIF-1α-deficient human vascular cells including vascular endothelial cells, vascular smooth muscle cells, and mesenchymal stem cells (MSCs), as a platform for discovering cell type-specific hypoxia-induced response mechanisms. Through comparative molecular profiling across cell types under normoxic and hypoxic conditions, we provide insight into the indispensable role of HIF-1α in the promotion of ischemic vascular regeneration. We found human MSCs to be the vascular cell type most susceptible to HIF-1α deficiency, and that transcriptional inactivation of ANKZF1, an effector of HIF-1α, impaired pro-angiogenic processes. Altogether, our findings deepen the understanding of HIF-1α in human angiogenesis and support further explorations of novel therapeutic strategies of vascular regeneration against ischemic damage.

DDX5 enhances HIF-1 activity by promoting the interaction of HIF-1α with HIF-1ß and recruiting the resulting heterodimer to its target gene loci.

BACKGROUND INFORMATION: Cancer cells acquire malignant characteristics and therapy resistance by employing the hypoxia-inducible factor 1 (HIF-1)-dependent adaptive response to hypoxic microenvironment in solid tumors. Since the underlying molecular mechanisms remain unclear, difficulties are associated with establishing effective therapeutic strategies. RESULTS: We herein identified DEAD-box helicase 5 (DDX5) as a novel activator of HIF-1 and found that it enhanced the heterodimer formation of HIF-1α and HIF-1ß and facilitated the recruitment of the resulting HIF-1 to its recognition sequence, hypoxia-response element (HRE), leading to the expression of a subset of cancer-related genes under hypoxia. CONCLUSIONS: This study reveals that the regulation of HIF-1 recruitment to HRE is an important regulatory step in the control of HIF-1 activity. SIGNIFICANCE: The present study provides novel insights for the development of strategies to inhibit the HIF-1-dependent expression of cancer-related genes.

Metastatic potentials classified with hypoxia-inducible factor 1 downstream genes in pan-cancer cell lines.

Hypoxia-inducible factor 1 (HIF1) is a transcription factor that is stabilized under hypoxia conditions via post-translational modifications. HIF1 regulates tumor malignancy and metastasis by gene transcriptions, such as Warburg effect and angiogenesis-related genes, in cancer cells. However, the HIF1 downstream genes show varied expressional patterns in different cancer types. Herein, we performed the hierarchical clustering based on the HIF1 downstream gene expression patterns using 1406 cancer cell lines crossing 30 types of cancer to understand the relationship between HIF1 downstream genes and the metastatic potential of cancer cell lines. Two types of cancers, including bone and breast cancers, were classified based on HIF1 downstream genes with significantly altered metastatic potentials. Furthermore, different HIF1 downstream gene subsets were extracted to discriminate each subtype for these cancer types. HIF1 downstream subtyping classification will help to understand the novel insight into tumor malignancy and metastasis in each cancer type.

A simplified protocol to induce hypoxia in a standard incubator: A focus on retinal cells.

Hypoxia chambers have traditionally been used to induce hypoxia in cell cultures. Cellular responses to hypoxia can also be mimicked with the use of chemicals such as cobalt chloride (CoCl2), which stabilizes hypoxia-inducible factor alpha-subunit proteins. In studies of ocular cells using primary cells and cell lines, such as Müller glial cell (MGC) lines, photoreceptor cell lines, retinal pigment epithelial (RPE) cell lines and retinoblastoma cell lines oxygen levels employed in hypoxia chambers range typically between 0.2% and 5% oxygen. For chemical induction of hypoxic response in these cells, the CoCl2 concentrations used typically range from 100 to 600 µM. Here, we describe simplified protocols for stabilizing cellular hypoxia-inducible factor-1α (HIF-1α) in cell culture using either a hypoxia chamber or CoCl2. In addition, we also provide a detailed methodology to confirm hypoxia induction by the assessment of protein levels of HIF-1α, which accumulates in response to hypoxic conditions. Furthermore, we provide a summary of conditions applied in previous studies of ocular cells.

An extra-erythrocyte role of haemoglobin body in chondrocyte hypoxia adaption.

Although haemoglobin is a known carrier of oxygen in erythrocytes that functions to transport oxygen over a long range, its physiological roles outside erythrocytes are largely elusive1,2. Here we found that chondrocytes produced massive amounts of haemoglobin to form eosin-positive bodies in their cytoplasm. The haemoglobin body (Hedy) is a membraneless condensate characterized by phase separation. Production of haemoglobin in chondrocytes is controlled by hypoxia and is dependent on KLF1 rather than the HIF1/2α pathway. Deletion of haemoglobin in chondrocytes leads to Hedy loss along with severe hypoxia, enhanced glycolysis and extensive cell death in the centre of cartilaginous tissue, which is attributed to the loss of the Hedy-controlled oxygen supply under hypoxic conditions. These results demonstrate an extra-erythrocyte role of haemoglobin in chondrocytes, and uncover a heretofore unrecognized mechanism in which chondrocytes survive a hypoxic environment through Hedy.

CopA3 treatment suppressed multidrug resistivity in HCT-116 cell line by p53-induced degradation of hypoxia-inducible factor 1α.

The major reason for multidrug resistance is the failure of chemotherapy in many tumors, including colon cancer. Hypoxia-inducible factor (HIF)-1α is a crucial transcription factor that simulates multiple cellular response to hypoxia. HIF-1α has been known to play a vital role towards tumor resistance; however, its mechanism of action is still not fully elucidated. N this study, we found that HIF-1α remarkably modulated drug resistance-associated proteins upon CopA3 peptide treatment against colon cancer cells. Abnormal rates of tumor growth along with high metastatic potential lacks the susceptibility towards cellular signals is a key characteristic in many tumor types. Moreover, in growing tumors, cells are exposed to insufficient nutrient supply and low oxygen availability. These stress force them to switch into adaptable and aggressive phenotypes. Our study investigated the interaction of HIF-1α and MDR gene association upon CopA3 treatment in the tumor microenvironment. We demonstrate that the multidrug resistance gene is associated with tumor resistance to chemotherapeutics, which upon CopA3 treatment promotes p53 activation and proteasomal degradation of HIF-1α, effecting the angiogenesis response to hypoxia. p53 downregulation augments HIF-1-dependent transcriptional activation of VEGF in response to oxygen deprivation.

Intracellular energy production and distribution in hypoxia.

The hydrolysis of ATP is the primary source of metabolic energy for eukaryotic cells. Under physiological conditions, cells generally produce more than sufficient levels of ATP to fuel the active biological processes necessary to maintain homeostasis. However, mechanisms underpinning the distribution of ATP to subcellular microenvironments with high local demand remain poorly understood. Intracellular distribution of ATP in normal physiological conditions has been proposed to rely on passive diffusion across concentration gradients generated by ATP producing systems such as the mitochondria and the glycolytic pathway. However, subcellular microenvironments can develop with ATP deficiency due to increases in local ATP consumption. Alternatively, ATP production can be reduced during bioenergetic stress during hypoxia. Mammalian cells therefore need to have the capacity to alter their metabolism and energy distribution strategies to compensate for local ATP deficits while also controlling ATP production. It is highly likely that satisfying the bioenergetic requirements of the cell involves the regulated distribution of ATP producing systems to areas of high ATP demand within the cell. Recently, the distribution (both spatially and temporally) of ATP-producing systems has become an area of intense investigation. Here, we review what is known (and unknown) about intracellular energy production and distribution and explore potential mechanisms through which this targeted distribution can be altered in hypoxia, with the aim of stimulating investigation in this important, yet poorly understood field of research.

A transient increase of HIF-1α during the G1 phase (G1-HIF) ensures cell survival under nutritional stress.

The family of hypoxia-inducible transcription factors (HIF) is activated to adapt cells to low oxygen conditions, but is also known to regulate some biological processes under normoxic conditions. Here we show that HIF-1α protein levels transiently increase during the G1 phase of the cell cycle (designated as G1-HIF) in an AMP-activated protein kinase (AMPK)-dependent manner. The transient elimination of G1-HIF by a degron system revealed its contribution to cell survival under unfavorable metabolic conditions. Indeed, G1-HIF plays a key role in the cell cycle-dependent expression of genes encoding metabolic regulators and the maintenance of mTOR activity under conditions of nutrient deprivation. Accordingly, transient elimination of G1-HIF led to a significant reduction in the concentration of key proteinogenic amino acids and carbohydrates. These data indicate that G1-HIF acts as a cell cycle-dependent surveillance factor that prevents the onset of starvation-induced apoptosis.

Hypoxia promotes conversion to a stem cell phenotype in prostate cancer cells by activating HIF-1a/Notch1 signaling pathway

Purpose The hypoxic tumor microenvironment and the maintenance of stem cells are relevant to the malignancy of prostate cancer (PCa). However, whether HIF-1α in the hypoxic microenvironment mediates the transformation of prostate cancer to a stem cell phenotype and the mechanism have not been elucidated.Materials and methods Prostate cancer stem cells (PCSCs) from PC-3 cell lines were examined for the expression of CD44, CD133, ALDH1, HIF-1α, Notch1, and HES1. We observed the effect of knockdown HIF-1α in vitro and mice models and evaluated the impact of HIF-1α on the Notch1 pathway as well as stem cell dedifferentiation. The effects on sphere formation, cell proliferation, apoptosis, cell cycle, and invasive metastasis were evaluated. Results In our study, hypoxia upregulated HIF-1α expression and induced a stem cell phenotype through activation of the Notch1 pathway, leading to enhanced proliferation, invasion, and migration of PCa PC-3 cells. The knockdown of HIF-1α significantly inhibited cell dedifferentiation and the ability to proliferate, invade and metastasize. However, the inhibitory effect of knocking down HIF-1α was reversed by Jagged1, an activator of the Notch1 pathway. These findings were further confirmed in vivo, where hypoxia could enhance the tumorigenicity of xenograft tumors by upregulating the expression of HIF-1α to activate the Notch1 pathway. In addition, the expression of HIF-1α and Notch1 was significantly increased in human PCa tissues, and high expression of HIF-1α correlated with the malignancy of PCa. Conclusion In a hypoxic environment, HIF-1α promotes PCa cell dedifferentiation to stem-like cell phenotypes by activating the Notch1 pathway and enhancing the proliferation and invasive capacity of PC-3 cells (AU)

Role of KLF4/NDRG1/DRP1 axis in hypoxia-induced pulmonary hypertension.

N-myc downstream regulated gene 1 (NDRG1) has recently drawn increasing attention because of its involvement in angiogenesis, cell proliferation, and differentiation. We used in vitro [human pulmonary artery smooth muscle cells (hPASMCs)] and in vivo (rat) models under hypoxic conditions and found a vital role of NDRG1 in reducing apoptosis and increasing proliferation and migration by overexpressing and knocking down NDRG1. We also proved that hypoxia induced the protein expression of dynamin-related protein 1 (DRP1) and stimulated The phosphatidylinositol-3-kinase (PI3K)/ Protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathways, and these effects were reversed by NDRG1 knockdown. The relationship between NDRG1 and DRP1 and the PI3K/Akt/mTOR pathway was further evaluated by adding mdivi-1 (DRP1 inhibitor) or LY294002 (PI3K inhibitor). NDRG1 was found to regulate the proliferation, apoptosis, and migration of hypoxia-treated hPASMCs via DRP1 and PI3K/Akt/mTOR signaling pathways. We explored the upstream regulators of NDRG1 using in vivo and in vitro hypoxia models. Hypoxia was found to upregulate and downregulate KLF transcription factor 4 (KLF4) protein expression in the cytoplasm and nucleus, respectively. Further, we showed that KLF4 regulated the proliferation and migration of hypoxia-treated hPASMCs via NDRG1. These results indicated a link between KLF4, NDRG1, and DRP1 for the first time, providing new ideas for treating hypoxic pulmonary hypertension.

Enhancement of Collagen-I Levels in Human Gingival Fibroblasts by Small Molecule Activation of HIF-1α.

Collagen is the most abundant protein in various mammalian tissues and has an essential role in various cellular processes. Collagen is necessary for food-related biotechnological applications such as cultivated meat, medical engineering, and cosmetics. High-yield expression of natural collagen from mammalian cells is challenging and not cost-effective. Thus, external collagen is obtained primarily from animal tissues. Under cellular hypoxia, overactivation of the transcription factor hypoxia-inducible factor (HIF) was shown to correlate with enhanced accumulation of collagen. Herein, we showed that the small molecule ML228, a known molecular activator of HIF, enhances the accumulation of collagen type-I in human fibroblast cells. We report an increase in collagen levels by 2.33 ± 0.33 when fibroblasts were incubated with 5 µM of ML228. Our experimental results demonstrated, for the first time, that external modulation of the hypoxia biological pathway can boost collagen levels in mammalian cells. Our findings pave the way for enhancing natural collagen production in mammals by altering cellular signaling pathways.

Hypoxia activates the PI3K/AKT/HIF-1α pathway to promote the anti-inflammatory effect of adipose mesenchymal stem cells.

This study aimed to investigate the effect of hypoxia on the anti-inflammatory effect of adipose-derived mesenchymal stem cells (AMSCs) in vitro and its possible mechanism. AMSCs were cultured in vitro in a hypoxic environment with 3% O2, and a normoxic (21% O2) environment was used as the control. The cells were identified by in vitro adipogenic and osteogenic differentiation and cell surface antigen detection, and the cell viability were detected. The effect of hypoxic AMSCs on macrophage inflammation was analyzed by co-culture. The results showed that under hypoxia, AMSCs had better viability, significantly downregulated the expression of inflammatory factors, alleviated macrophage inflammation, and activated the PI3K/AKT/HIF-1α pathway.

Generation and Characterization of CYP2E1-Overexpressing HepG2 Cells to Study the Role of CYP2E1 in Hepatic Hypoxia-Reoxygenation Injury.

The mechanisms of hepatic ischemia/reperfusion (I/R) injury, which occurs during liver transplantation or surgery, are poorly understood. The purpose of the current study was to generate and characterize a HepG2 cell line with a stable overexpression of CYP2E1 to investigate the role of the enzyme in hypoxia/reperfusion (H/R) injury in an ex vivo setting. GFP-tagged CYP2E1 and control clones were developed, and their gene expression and protein levels of GFP and CYP2E1 were determined using RT-PCR and ELISA/Western blot analysis, respectively. Additionally, the CYP2E1 catalytic activity was determined by UPLC-MS/MS analysis of 6-hydroxychlorzoxazone formed from the chlorzoxazone substrate. The CYP2E1 and control clones were subjected to hypoxia (10 h) and reoxygenation (0.5 h), and cell death and reactive oxygen species (ROS) generation were quantitated using LDH and flow cytometry, respectively. Compared with the control clone, the selected CYP2E1 clone showed a 720-fold increase in CYP2E1 expression and a prominent band in the western blot analysis, which was associated with a 150-fold increase in CYP2E1 catalytic activity. The CYP2E1 clone produced 2.3-fold more ROS and 1.9-fold more cell death in the H/R model. It is concluded that the constitutive CYP2E1 in the liver may play a detrimental role in hepatic I/R injury.

Hypoxia-induced reprogramming of glucose-dependent metabolic pathways maintains the stemness of human bone marrow-derived endothelial progenitor cells.

The benefits of hypoxia for maintaining the stemness of cultured human bone marrow-derived endothelial progenitor cells (BM EPCs) have previously been demonstrated but the mechanisms responsible remain unclear. Growing evidences suggest that cellular metabolism plays an important role in regulating stem cell fate and self-renewal. Here we aimed to detect the changes of glucose metabolism and to explore its role on maintaining the stemness of BM EPCs under hypoxia. We identified the metabolic status of BM EPCs by using extracellular flux analysis, LC-MS/MS, and 13C tracing HPLC-QE-MS, and found that hypoxia induced glucose metabolic reprogramming, which manifested as increased glycolysis and pentose phosphate pathway (PPP), decreased tricarboxylic acid (TCA) and mitochondrial respiration. We further pharmacologically altered the metabolic status of cells by employing various of inhibitors of key enzymes of glycolysis, PPP, TCA cycle and mitochondria electron transport chain (ETC). We found that inhibiting glycolysis or PPP impaired cell proliferation either under normoxia or hypoxia. On the contrary, inhibiting pyruvate oxidation, TCA or ETC promoted cell proliferation under normoxia mimicking hypoxic conditions. Moreover, promoting pyruvate oxidation reverses the maintenance effect of hypoxia on cell stemness. Taken together, our data suggest that hypoxia induced glucose metabolic reprogramming maintains the stemness of BM EPCs, and artificial manipulation of cell metabolism can be an effective way for regulating the stemness of BM EPCs, thereby improving the efficiency of cell expansion in vitro.

Photobiomodulation at 660 nm promotes collagen synthesis via downregulation of HIF-1α expression without photodamage in human scleral fibroblasts in vitro in a hypoxic environment.

PURPOSE: The increasing prevalence of myopia is a global public health issue. Because of the complexity of myopia pathogenesis, current control methods for myopia have great limitations. The aim of this study was to explore the effect of photobiomodulation (PBM) on human sclera fibroblasts (HSFs) under hypoxia, in the hope of providing new ideas for myopia prevention and control. METHODS: Hypoxic cell model was established at 0, 6, 12, and 24 h time points to simulate myopia microenvironment and explore the optimal time point. Control, hypoxia, hypoxia plus light, and normal plus light cell models were set up for the experiments, and cells were incubated for 24 or 48 h after PBM (660 nm, 5 J/cm2), followed by evaluation of hypoxia-inducible factor 1α (HIF-1α) and collagen I a1 (COL1A1) proteins using Western blotting and immunofluorescence, and photo damage was detected by CCK-8, scratch test, and flow cytometry assays. We also used transfection technology to further elucidate the regulatory mechanism. RESULTS: The change of target proteins is most obvious when hypoxia lasts for 24 h (p < 0.01). PBM at 660 nm increased extracellular collagen content (p < 0.001) and downregulated expression of HIF-1α (p < 0.05). This treatment did not affect the migration and proliferation of cells (p > 0.05), and effectively inhibited apoptosis under hypoxia (p < 0.0001). After overexpression of HIF-1α, the effect of PBM was attenuated (p > 0.05). CONCLUSIONS: Photobiomodulation at 660 nm promotes collagen synthesis via downregulation of HIF-1α expression without photodamage.