Hypoxia Inducible Factor-1α in Pulmonary Fibrosis: A Promising Therapeutic Target

Pulmonary fibrosis (PF) is a chronic and progressive disease depicted by excessive scarring, which leads to increased tissue stiffness and loss of lung function. The condition is caused by small injuries to the alveolar epithelium, consequential in the formation of fibroblasts, myofibroblasts, and fibroblastic foci areas within the lung tissue. These cells deposit an excessive amount of collagen-rich extracellular matrix (ECM). Hypoxia, along with its transcription factor known as hypoxia-inducible factor-1 alpha (HIF-1α), activates various signaling pathways that can promote to the progression of PF by promoting myofibroblast differentiation and ECM accumulation. HIF-1α plays a significant role in sustaining inflammatory lung micro-injury, stimulating growth factors, and contributing to PF pathogenesis. Therefore, targeting HIF-1α could be a promising approach to inhibit the progression of PF. This review article discusses the various signaling pathways, excessive ECM formation, and related growth factors involved in HIF-1α regulation in PF, as well as explores the potential use of HIF-1α inhibitors to mitigate PF.

Introduction

Pulmonary fibrosis is a progressive, chronic, and fatal disease characterized by multiple micro-injuries to the alveolar epithelium, fibroblast proliferation, excessive deposition of ECM, tissue stiffening, destruction of the alveolar architecture, and a relentless decline in lung function [1]–[3]. Researchers have identified the cells, inflammatory mediators, and signaling pathways responsible for the growth of PF over the past decade [4], [5]. Alveolar epithelial cells (AECs) undergo epithelial-mesenchymal transition (EMT), leading to the origin of myofibroblasts, the major cells responsible for the accumulation of collagen-rich ECM in the lung [6].

Hypoxic tissue microenvironment induces the formation of HIF-1α, a master transcription factor for oxygen homeostasis [7], [8]. In normoxia, HIF-1α is destabilized by hydroxylation followed by proteasomal degradation [9]. However, HIF-1α can be stabilized under hypoxic conditions and trans-activate the target genes.

Several investigations have proposed that HIF-1α is the prominent factor associated with the pathogenesis and progression of PF through the transformation of AECs to fibroblasts [10]. HIF-1α signaling stimulates the EMT and could induce the migration of epithelial cells, thus up-regulating the EMT in PF [11], [12]. Reactive oxygen species and HIF-1α signaling are upstream of TGFβ1 production in the hypoxic milieu of tissue fibrosis, and TGFβ1 increases ROS production while stabilizing HIF-1α in the fibrotic tissue, indicating the essential role of HIF-1α signaling in PF [13].

Signaling pathways, such as TGFβ1, VEGF, MAPK, Snail, β-catenin, and SMAD, up-regulate the expression of HIF-1α. TGFβ1 is a key inducer of lung fibrosis and enhances the stability of HIF-1α in the fibrotic tissue-derived fibroblasts. The activation of HIF-1α coupled with the sustained TGFβ1 signaling suggests a positive feedback-loop of HIF-1α/TGFβ1 in the progression of PF [14]. HIF-1α expression can be stabilized through its protective effect, due to the formation of a complex with SMAD, triggering the formation of alpha-smooth muscle actin (α-SMA) in fibroblasts, thereby directly up-regulating myofibroblast differentiation [15]. Moreover, chronic inflammation and hypoxia are risk factors for PF, as inflammatory interferon activates the HIF-1α-mediated PI3K/Akt pathway promoting EMT [16], [17]. Inhibition of HIF-1α is therefore a rational strategy for novel therapeutic development, given the lack of effective therapies currently available for PF. This review appraises some signaling pathways, related growth factors that contribute to HIF-1α regulation, and therapeutic strategies to inhibit HIF-1α activity in PF.

HIF-1α Regulates Epithelial-Mesenchymal Transition Through the Snail and β-Catenin Pathways

EMT is a process in which specialized epithelial cells undergo biochemical changes, transforming into a type of cell with invasive properties. This transformation results in increased opposition to cell death, higher production of ECM, loss of cell-cell adhesion and polarity, and the acquisition of mesenchymal cell characteristics. These characteristics include elevated levels of N-cadherin and α-SMA, which are markers of mesenchymal cells and contribute to migratory capacity [18].

In the setting of lung function, alveolar epithelial cells can undergo EMT, leading to increased deposition of ECM and promoting PF [19].

Specific cellular factors perform a role in regulating EMT. The transcription factor Snail suppresses adhesion molecules ZO-1, claudin-1, and E-cadherin in epithelial cells, influencing EMT [20]. Additionally, the protein complex β-catenin is involved in the progression of EMT, acting as a mediator of Wnt signaling that affects cell proliferation and differentiation [21]. Under normal conditions, β-catenin helps link E-cadherin to actin; however, during EMT, it serves as a transcriptional co-activator, promoting the transcription of genes that induce EMT [22].

Furthermore, HIF-1α plays a role in inducing EMT by regulating the expression of Snail and E-cadherin. There is a positive correlation between signaling pathways involving Snail, β-catenin, and HIF-1α, contributing to the transformation of lung AECs into mesenchymal cells in the context of PF [23], [24] (Fig. 1).

Fig. 1. HIF-1α induces EMT in the lung through Snail and β-catenin signaling. Paraquat creates a hypoxic environment and increases the level of HIF-1α in the lung, leading to an increase in Snail and β-catenin, which can promote the transcription of genes that induce EMT and contribute to the development of pulmonary fibrosis.

HIF-1α and TGFβ1/VEGF Signaling Pathways

The fibrotic process is associated with hypoxia due to the loss of endothelial cells and the rarefaction of capillaries caused by Endothelial-mesenchymal transition (EndoMT), a process in which endothelial cells adopting a mesenchymal (fibroblast-like) phenotype, allowing these cells to migrate and acquire invasive properties. Inflammation or chronic diseases can activate EndoMT and EMT processes [25].

Studies on idiopathic pulmonary fibrosis (IPF) showed that the fibrotic process correlates with the HIF-1α target genes such as tissue inhibitor of metalloproteinase-1 (TIMP-1), plasminogen activator inhibitor-1 (PAI-1), and connective tissue growth factor (CTGF) [26]. TGFβ1 and hypoxia signaling appear to undergo mutual interactions since the major TGFβ1-responsive transcription factor Smad-3 can be up-regulated via hypoxia and vice versa [27]. Therefore, TGFβ1 and hypoxic milieu in injured tissues are drivers of EMT in PF.

VEGF is a potent angiogenic factor known to date; it inhibits cell apoptosis and induces cell proliferation, migration, differentiation, and vascular permeability. HIF-1α promotes VEGF expression to regenerate vessels, linked to the formation of fibroblast and EMT [28]. Diminished HIF-1α in epithelial cells reduces EMT, and the targeted deletion of HIF-1α in epithelial cells reduces interstitial fibrosis. These research outcomes state that hypoxia is prominent in injured tissue and can trigger angiogenesis via HIF-1α-driven VEGF. Thus, VEGF is thought to contribute to the pathogenesis of lung fibrosis, and its positive cells are abundantly present in fibrotic lungs, mostly type-2 pneumocytes and myofibroblasts. VEGF activity in type-2 pneumocytes and myofibroblasts promotes mast cells and macrophage aggregation in the fibrotic lung [29]. Moreover, VEGF is an indirect leukocyte migrating factor related to the development of MCP-1, IL-8, and ECM synthesis [30]. Therefore, systemic inhibition of VEGF/VEGFR in severe fibrotic lung regions may have protective effects against the progression of PF.

Phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) pathways are central downstream signaling pathways of VEGF, which can activate these signaling pathways to modulate vasoactive function and angiogenic response. Besides hypoxic signaling, the MAPK and PI3K/Akt pathways are common in regulating HIF-1α activity [31]. PI3K/Akt directly participates in the formation of PF or cooperates with HIF-1α to promote the expansion of PF. Accordingly, HIF-1α transcriptional activity could be inhibited by blocking MAPK and PI3K/Akt signals, thereby stopping the progression of PF. Taken together, HIF-1α, TGFβ1, VEGF, MAPK, and PI3K/Akt signaling paths have a close association with the progression of PF. Thus, HIF-1α forms a positive regulatory feedback loop with VEGF via the activation of MAPK and PI3K/Akt signaling (Fig. 2).

Fig. 2. HIF-1α and VEGF create a positive feedback loop by activating PI3K and MAPK signaling. Growth factors and hypoxic signaling activate VEGF, which in turn triggers the PI3K/Akt and MAPK signaling to regulate HIF-1α activity.

HIF-1α Induces the Buildup of Extracellular Matrix in Pulmonary Fibrosis

The ECM provides mechanical support to the lungs and is made up of fibrous proteins (collagens and elastin), adhesive proteins (fibronectin and tenascin), and glycosaminoglycans. Collagen fibers are the most common components of the pulmonary ECM, contributing to the lung’s shape, compliance, and elasticity. The accumulation of ECM leads to the progression of fibrosis, which is a result of acute lung injury (ALI). ALI triggers inflammatory mediators and leads to inflammation, playing a central role in the development of PF. Acute lung inflammation is characterized by diffuse alveolar edema and systemic inflammation, leading to hypoxia, progressive fibrosis, and pulmonary hypertension (PH).

HIF-1α contributes to the increased expression of genes responsible for various ECM proteins in lung fibroblasts. It induces collagen hydroxylation and the biosynthesis of collagen in the hypoxic environment by directly activating the transcription of collagen prolyl-4-hydroxylase [32]. HIF-1α also up-regulates the enzyme lysyl oxidase (LOX), which initiates cross-linkage in elastin and collagen, contributing to the remodeling of the ECM [33]. Therefore, HIF-1α mediates LOX for the deposition of ECM in the lung, leading to the persistence of PF by promoting the establishment of elastin and collagen-rich ECM.

A hallmark of PF is the presence of myofibroblasts in bunches known as fibroblastic foci and the deposition of ECM in the interstitium of the lung. Hypoxia-induced cellular oxidative stress causes multiple micro-injuries to alveolar epithelial cells, leading to the release of profibrotic factors and the proliferation and activation of fibroblasts [34]. The continued proliferation of fibroblasts and myofibroblasts, along with ECM deposition, damages the lung parenchyma and the epithelial barrier, further influencing cell behavior and accelerating the progression of PF. Abnormal ECM in the lung tissue alters the biochemical and mechanical properties, establishing a hypoxic environment.

These findings underscore that HIF-1α activation triggers ECM production, leading to progressive PF, and offer new insights for identifying treatment targets (Fig. 3).

Fig. 3. HIF-1α is implicated in the formation of ECM and progressive PF. Inflammatory signaling and TGF-β1 activity in the lung create a hypoxic milieu, inducing the persistence of HIF-1α, which triggers ECM formation and progressive PF.

Emphasizing Therapeutic Strategies Targeting HIF-1α

Previous investigations have shown that HIF-1α is involved in the progression of PF and pulmonary hypertension (PH). Targeting HIF-1α could potentially be an effective treatment approach for PF. Strategies that focus on addressing hypoxia may involve inhibiting the hypoxia signaling pathway through HIF-1α inhibition (see Table 1).

Table I. Therapeutic Approaches Concerning HIF-1α

Category	Drug	Reference
HIF-1α inhibitors	YC-1	[36]
	Topotecan	[37]
	Digoxin	[38]
HDAC inhibitor	Valproic acid	[40]
VEGF and HIF-1α inhibitor	Sulforaphane	[42]

Hypoxic prodrugs can be activated in low-oxygen environments, delivering the active mediator to hypoxic tissues through prodrug reduction by reductases [35]. This approach could help reduce off-target effects by targeting the active drug to tissue hypoxia and inhibiting HIF-1α. Gene therapy targeting HIF-1α is an effective treatment, and liposome-based drug delivery can be used for this purpose. For example, the liposome polyethylene glycol polymer has been successfully used to deliver doxorubicin combined with antisense oligonucleotide targeted to the mRNA of HIF-1α. In addition, the HIF-1α inhibitor YC-1 [3-(50-hydroxymethyl-20-furyl)-1-benzyl indazole] can reduce ECM accumulation [36]. Studies have shown that topotecan can block luciferase activity and VEGF production by reducing HIF-1α [37]. In vivo and in vitro studies have demonstrated that digoxin effectively reduces HIF-1α activity in PC3 cells [38]. Epigenetic therapy, such as DNMT inhibitors, HDAC inhibitors, and microRNA-targeted drugs, can also be used to block HIF-1α [39]. Similarly, valproic acid and other HDAC inhibitors could suppress angiogenesis by decreasing the expression of VEGF and HIF-1α [40]. Inhibition of the fibroblast HIF-1α/PDK1 (pyruvate dehydrogenase kinase1) axis by HIF-1α gene deletion or inhibition of PDK1 attenuates bleomycin-induced PF [41]. The research suggests that sulforaphane can inhibit the HIF-1α activity and the hypoxia-induced expression of VEGF [42]. This inhibition works by destabilizing the HIF-1α protein and preventing the activation of its target genes. Understanding the connection of HIF-1α in the advancement of lung fibrosis could offer new treatment possibilities by targeting the feedback-loop signaling pathway of HIF-1α and VEGF. Since HIF-1α is linked with certain inflammatory pathways, blocking inflammatory mediators could be a logical approach to treatment. Deleting fibroblast HIF-1α could be an anti-fibrotic strategy by decreasing the recruitment of inflammatory cells into fibrotic tissue [43]. Additionally, the combined effect of HIF-1α and TGFβ1 in the advancement of PF makes this axis a potential treatment target.

Inhibiting PI3K/Akt signaling could prevent fibroblast-to-myofibroblast differentiation (FMD), consequently reducing HIF-1α activity and improving PF [44], [45]. Suppressing Akt phosphorylation by inhibiting PI3K activity and enhancing PTEN activity has been found to reduce PF. Although the exact mechanism of HIF-1α regulation is not fully understood, research indicates that treatment strategies targeting HIF-1α could be crucial in preventing the progression of PF.

Conclusion and Future Prospects

Extensive evidence suggests that HIF-1α plays an important role in the development of PF, and its over-expression has serious implications for lung fibrosis. Continued exposure to HIF-1α is detrimental to the lung’s AECs and contributes to irreversible fibrosis and disease progression, ultimately leading to organ failure and high morbidity and mortality rates. Thus, targeting HIF-1α and its associated pathways is crucial to halt PF progression. Understanding the role of HIF-1α in PF offers new insights for treatment. HIF-1α, along with TGFβ1, VEGF, β-catenin, Snail, PI3K/Akt, and MAPK signaling pathways, is closely linked to PF. HIF-1α and VEGF create a positive feedback loop through the MAPK and PI3K/Akt signaling, making this feedback-loop signaling pathway a potential therapeutic strategy. Targeting genes controlled by HIF-1α could effectively inhibit PF progression. While the precise mechanism of HIF-1α regulation is not fully understood, research suggests that therapeutic approaches targeting HIF-1α could significantly impede PF progression.