Hypoxia-induced signaling in Gut/Liver pathobiology

Abstract

Oxygen (O₂) is essential for cellular metabolism and biochemical reactions. When the demand for O₂ exceeds the supply, hypoxia occurs. Hypoxia-inducible factors (HIFs) are essential to activate adaptive and survival responses following hypoxic stress. In the gut (intestines) and liver, oxygen gradients or “physiologic hypoxia” are necessary to maintain normal homeostasis. While physiologic hypoxia is beneficial and aids in normal functions, pathological hypoxia is harmful as it exacerbates inflammatory responses, tissue dysfunction, and is a hallmark of many cancers. In this review, we discuss the role of gut and liver hypoxia-induced signaling, primarily focusing on HIFs, in the physiology and pathobiology of gut and liver diseases. Additionally, we examine the function of HIFs in various cell types during gut and liver diseases, beyond intestinal epithelial and hepatocyte HIFs. This review highlights the importance of understanding hypoxia-induced signaling in the pathogenesis of gut and liver diseases and emphasizes the potential of HIFs as therapeutic targets.

1. INTRODUCTION:

Molecular oxygen (O₂) is vital for life. Physiological oxygen gradients or partial pressure of oxygen (pO2) varies due to dynamic tissue/organ blood perfusion rates playing important roles in mammalian physiology. Physiological pO2 for lung, brain, intestine, and liver are 110, 35, 58 and 41 mmHg, respectively (1). However, when oxygen gradients fall below the normal physiological range, organisms need to adapt to support key cellular activities until oxygen homeostasis is restored. Chronic tissue oxygen deprivation (or hypoxia) can have deleterious consequences on organismal homeostasis.

Hypoxia-inducible factors (HIFs) were discovered in the 1990s and are crucial transcription factors essential for adapting to hypoxia via transcriptional reprogramming of critical genes in cellular energetics, oxygen delivery, and cell survival. The discovery of HIFs and their regulation by Kaelin, Ratcliffe, and Semenza provided a framework for our understanding of cellular O₂ sensing (2–4). The groundbreaking discoveries of HIFs also led to new avenues of investigation and demonstrated that HIFs play important roles in physiology and pathology, such as inflammation and cancer (5). HIFs belong to the PER-ARNT-SIM (PAS) subfamily of the basic helix-loop-helix (bHLH) transcription factors, and they are evolutionarily conserved in all metazoans (6). Vertebrates express three isoforms of O₂ sensitive HIFα-subunits, namely (HIF1α, HIF2α, and HIF3α) and a constitutively expressed HIFβ subunit (Arnt, aryl hydrocarbon receptor nuclear translocator). Under normoxia, prolyl hydroxylase enzymes (PHDs) use O₂ as substrate and hydroxylate two conserved proline residues on HIFα-subunits. Hydroxylated HIFα proteins are recognized by an E3 ubiquitin ligase, Von Hippel-Lindau tumor suppressor protein (VHL) which target HIFs for proteasomal degradation (4; 7). Additionally, Factor-Inhibiting HIF-1 (FIH-1) enzyme hydroxylates a conserved asparagine residue in the C-TAD domain, blocking the association of HIFα with the p300 coactivator protein (8). Thus, HIFs are tightly regulated by prolyl and asparaginyl hydroxylation under normoxia. However, under hypoxia, PHDs and FIH-1 can no longer utilize O₂ as substrates leading to HIFs stabilization. HIFα subunit dimerize with HIFβ, translocate to the nucleus, and bind to hypoxia response elements (HRE) on promoters of target genes (Figure 1A).

Figure 1: HIF signaling and oxygen dynamics in the intestine and liver.

A) HIF signaling pathway: In normoxia, in the presence of O₂, α-ketoglutarate (α-KG), and Fe²⁺, PHDs and FIH-1 enzymes hydroxylate HIF1/2α enabling recognition and binding by von-Hippel Lindau tumor suppressor (VHL). This results in ubiquitination and proteasomal degradation. In hypoxia, due to lack of O₂, PHDs and FIH-1 enzymes are rendered inactive stabilizing HIF1/2α. HIFs binds to HIF1β to drive transcription of target genes. B) Gut oxygen dynamics: The small intestinal and colonic intestinal tract respectively exhibit a pO2 gradient of ~58 and ~42 mmHg pO2 in highly vascularized submucosa to ~22 and ~10 mmHg at the villus tip attributed to combination of complex intestinal vascular system and the presence of commensal microbes in an anoxic lumen. C) Liver oxygen dynamics: The hepatic acinus exhibit a pO2 gradient, with the periportal region being highly oxygenated (pO2 is ~65 mm Hg), and the pericentral region exhibit pO2 is ~35 mm Hg. Abbreviations: a-KG, a-ketoglutarate; Fe²⁺, iron; FIH, factor-inhibiting HIF; HIF, hypoxia-inducible factor; PHD, prolyl hydroxylase domain; pO2, partial pressure of oxygen; Ub, ubiquitination; VHL, von Hippel-Lindau tumor suppressor.

PHDs are evolutionarily conserved subfamily of dioxygenases and mammals contain three isoforms (PHD1–3) having overlapping and differential tissue expressions (9). PHDs have K_m values of 230–250 μM for O₂ while FIH have K_m values of 90 μM, thus making sensitive to large O₂ variations (10). While the PHD-catalyzed hydroxylation reactions and subsequent HIF activation are reliant on O₂ levels, HIFs are also regulated by PHDs in an oxygen-independent manner. For instance, PHD activity is influenced by cofactors such as α-ketoglutarate (αKG), Fe²⁺, ascorbate, succinate, and mitochondrial reactive oxygen species (11). Mutations in mitochondrial enzymes such as isocitrate dehydrogenases, succinate dehydrogenase can reduce αKG levels and increase succinate respectively leading to PHDs inhibition and HIFs activation (12). Similarly, decreased intracellular Fe²⁺ levels activate HIFs as PHDs require Fe²⁺ for enzymatic activity (13). Additionally, reactive oxygen species (14), inflammatory cytokines via NF-kB (15), oncogenic signaling like PI3K/Akt/mTORC1 (16) contribute to HIFs induction. Notably, the downstream target genes regulated by HIF signaling can differ depending on the cellular stimuli that activate the pathway. Furthermore, HIF1α and HIF2α bind to the same canonical hypoxia response elements (HREs) but have nonredundant functions in regulating distinct downstream targets. These differences are primarily attributed to differential tissue expression, differential regulation by PHDs, diverse interacting partners, and temporal activation kinetics (17). This review aims to outline our current understanding of hypoxia-induced HIF signaling in the context of gut and liver homeostasis. We will also focus on how deregulation of HIF signaling contributes to gut and liver pathologies and how these pathways could be exploited as therapeutic targets.

2. OXYGEN DYNAMICS:

The development of sophisticated techniques over the last few decades have enabled measurement of tissue pO2 (18). Distinct gut and liver oxygen gradients influence physiological functions. Gut oxygen gradients are essential in barrier function and nutrient absorption (18), while liver oxygen gradients contribute to liver zonation and glucose metabolism (19).

2.1. Gut oxygen dynamics:

Intestinal epithelial cells maintain healthy barrier against intraluminal toxins, gut microbiota, absorption of macro- and micro-nutrients, electrolytes, water, and removal of waste. Additionally, the intestinal epithelium play a critical role in regulating whole body oxygenation by controlling iron absorption (13). This highly regenerative organ consists of a monolayer of epithelial cells covering an area of approximately 300–400 m² and completely renews every 3–5 days in a tightly controlled process. The adult intestinal epithelial lining is organized into well-defined structures known as the crypt-villus axis. The crypt forms the base and is composed of stem cells, while the villus are finger-like projections extending into the lumen and composed of differentiated mature cells (20). The intestinal epithelium display a unique oxygen gradient, whereby the subepithelial mucosa on basolateral surface is highly oxygenated while the luminal side is almost anoxic. Under steady-state physiological conditions, the small intestine and colon exhibit respective gradients of ~ 58 mmHg pO2 and ~42 mmHg pO2 in highly vascularized submucosa, ~22 mmHg and ~5–10 mmHg at the villus tip and <10 mmHg and <3 mmHg in the lumen (Figure 1B) (18). These differences in oxygen gradients along the crypt-villus axis are largely attributed to combination of complex intestinal vascular system affecting blood perfusion rates and the presence of large number of commensal microbes that exist in an anoxic lumen. Thus, mucosal epithelial cells always exist in nearly anoxic environment, referred to as “physiologic hypoxia”.

2.2. Liver oxygen dynamics:

Liver is a central metabolic hub whose functions include maintaining blood glucose levels, detoxification of xenobiotics, synthesizing several circulating systemic factors to maintain organismal homeostasis. Previously considered a homogenous organ, it is now known to be structurally and functionally heterogeneous. The hepatocytes are the major parenchymal cell type in the liver and are mainly postmitotic but have a robust regenerative capacity. Hepatocytes are arranged into hexagonal units called lobules, which form the structural unit of the liver. The functional unit of the liver is the hepatic acinus, which is supplied by a single terminal branch of the hepatic artery. The acinus is divided into three zones, with zone 1 located in the periportal areas surrounding the hepatic arteries and portal veins, zone 3 in the perivenous/pericentral areas surrounding the central vein, and zone 2 located in the intermediary or mid-lobular region between zones 1 and 3. Hepatic zones are characterized by distinct gene profiles and metabolic pathways. For instance, β-oxidation and lipid synthesis enzymes are expressed predominantly in periportal & pericentral regions respectively (21). Oxygen and nutrients are supplied to liver cells via two afferent vessels, the hepatic artery and portal vein, whereby blood enters the lobule and flow towards the central vein. Like, intestine, the hepatic acinus also display a unique oxygen gradient across the periportal & pericentral regions, with the periportal region (zone 1) proximal to the blood supply being highly oxygenated (pO2 is 60–65 mm Hg), while the pericentral region (zone 3) is relatively hypoxic (pO2 is 30–35 mm Hg) (Figure 1C). The liver expresses all HIFs isoforms & oxygen gradient across periportal/pericentral regions drive discrete transcriptional responses, modulate signaling pathways like Wnt/β-catenin, contributing to liver zonation (19). Additionally, hepatic oxygen dynamics are influenced by the fasting/feeding status (22). Thus, dynamic hepatic oxygen gradient allow spatially functional heterogeneity in the lobules/acini maintaining liver homeostasis.

3. HIFs IN GUT & LIVER PHYSIOLOGY:

As “physiologic hypoxia” is a salient feature of gut and liver, the role of HIFs in gut & liver physiology have been intensively interrogated.

3.1a. HIFs in gut cellular metabolism:

Glycolysis is a metabolic pathway that involves the breakdown of glucose to produce pyruvate. In tissues with sufficient oxygen concentration, pyruvate is subsequently oxidized via the TCA cycle and oxidative phosphorylation in the mitochondria to generate ATP. The best-known metabolic function of HIFs is regulating glycolysis (5). Additionally, hypoxia, through HIF-dependent and independent pathways, is central in regulating several metabolic pathways, including mitochondrial respiration and lipid metabolism (23). HIFs also interact with several nutrient sensing pathways, such as AMP-activated protein kinase, integrated stress response, and the mechanistic target of rapamycin complex 1 (mTORC1) to coordinate nutrient availability with growth. As HIFs influence metabolism, there has been efforts to decipher the role of HIF mediated cellular metabolism in maintaining normal gut and liver physiology. Interestingly, unlike whole body HIF1α and HIF2α null mice which are embryonic lethal (24), intestinal epithelial or hepatocyte specific HIF1α and HIF2α are dispensable for normal gut and liver development (25). In contrast, both intestinal and liver HIF signaling are critical to regulate systemic metabolism. Beneficial effects of gastric bypass surgery in glycemic control were attributed to increased intestinal glucose metabolism (26). Intestinal HIF2α was reported to be increased following vertical sleeve gastrectomy (VSG) and was found to critical to improve glucose tolerance (27). Moreover, small intestinal epithelial HIF2α is essential for increasing systemic ceramides regulating body weight and adiposity (28). Recent studies suggest that heteroceullar metabolic exchange between the different intestinal epithelial cells are essential to maintain homeostasis (29). Whether HIFs contribute to such metabolism to maintain intestinal homeostasis remains unknown.

3.1b. HIFs in liver cellular metabolism:

Liver is the principal organ regulating metabolism via insulin and metabolic hormones maintaining blood glucose homeostasis. In the fed state, liver converts glycolytic products to fatty acids, which can be stored or secreted as VLDL (very low density lipoprotein). In the fasted state, liver produces glucose via glycogenolysis and gluconeogenesis, and converts fatty acids into ketone bodies for utilization in extrahepatic tissues. HIFs play important roles in hepatic glucose metabolism (30). Hepatocyte specific disruption of HIF1α impaired gluconeogenesis during liver regeneration (31) and improved obesity induced glucose intolerance via reducing GLP-1 degradation (32). Hepatocyte HIF2α was shown to regulate insulin signaling, by augmenting insulin signaling through induction of insulin receptor substrate-2 (33; 34). Fasting/refeeding influence liver oxygen dynamics and transient hepatocyte HIF2α activation during refeeding was shown to abrogate postprandial glucagon signaling, thereby suppressing gluconeogenesis, and ameliorating hyperglycemia (22). Additionally, HIFs regulate glucose homeostasis through indirect mechanisms. Erythropoietin, a well-characterized HIF2α target gene (35) was shown to improve systemic glucose homeostasis via reducing adipose tissue inflammation thereby improving insulin sensitivity (36). Interestingly, Chuvash polycythemia patients who display increased HIF due to a homozygous mutation in the VHL have decreased serum glucose and insulin concentrations suggesting an important role of HIFs in glucose homeostasis (37). HIFs also modulate lipid metabolism by driving reductive carboxylation of glutamine-derived α-ketoglutarate (38). Indeed, hepatocyte specific activation of HIF1α (39) or HIF2α (40) regulate key genes involved in hepatic lipid uptake and synthesis thereby influencing hepatic lipid metabolism.

3.2. HIFs in gut and liver nutrient metabolism:

The intestine and liver are important sites for the entry and generation of key nutrients for systemic use. While it is well-established that hypoxia-inducible factors (HIFs) regulate transport of nutrients in the cancer setting (41) less is known about their role in the normal intestine and liver nutrient metabolism. Transcriptomics datasets suggest that HIFs regulate a large subset of nutrient transporters, with the best characterized nutrient that requires HIF signaling being iron (42). Intestine and liver regulate systemic iron metabolism by regulating iron absorption, storage, and release into circulation and iron homeostasis is critical for its function. While the intestinal epithelium absorbs dietary iron in its ferrous form through the apical iron transporter, Divalent metal transporter 1 (DMT1) and exports iron through ferroportin, the liver-derived hormone hepcidin regulates iron export by binding to ferroportin (43). In states of iron deficiency and hypoxia, intestinal HIF2α regulates the expression of DMT1 and ferroportin (13; 44), while hepatocyte HIF2α regulate the expression of hepcidin through erythropoietin (45). Recent studies by Schwartz and colleagues demonstrated crosstalk between the hepatic hepcidin/intestinal HIF2α, regulating systemic iron metabolism (46).

3.3. HIFs in Gut and liver barrier function:

The intestinal epithelium and its tight junction proteins, mucins, and antimicrobial peptides are crucial in maintaining intestinal homeostasis. Tight junction and adherens proteins, including claudins, occludins, and desmosomes, constitute the intestinal apical junctional complex (47). HIFs regulate and increase the expression of several proteins and pathways essential for maintaining junctional fidelity as reviewed in (18). Short-chain fatty acids such as butyrate increase oxygen consumption and activate HIF1α to maintain intestinal barrier integrity (48). Adenosine signaling is known to influence barrier function and HIF1α increases adenosine availability by regulating CD73 expression (49). HIFs are also critical in intestinal wound repair responses by modulating integrin α2 and α6 levels (50). However, chronic activation of HIF2α expression increases caveolin-1 expression, resulting in rapid turnover of occludins and compromised barrier integrity (51). HIFs also regulate the secretion of antimicrobial peptides, mucins, and trefoil factors (TFFs). Muc2 is the predominant mucin expressed in the intestine, and HIF2α induces its expression (52), while Muc3 and Muc5ac are HIF1α targets (53). Likewise, TFF3 was shown to be induced by HIF1α (54). Lastly, antimicrobial peptides Human β-defensin-1 were found to be dependent on HIF1α in mice and human colonic tissues (55). Liver sinusoidal endothelial cells (LSECs) play a crucial role in maintaining liver barrier and homeostasis, acting as gatekeeper cells between the liver sinusoids and parenchyma (21). LSECs also participate in liver regeneration after injury. Although the role of HIFs in liver barrier function has not been extensively investigated unlike intestines, studies have shown that HIFs participate in liver barrier function. LSECs fenestration and proliferation was shown to be dependent on HIF1α/VEGF axis (56). Collectively, intestinal epithelial and hepatocyte HIFs under “physiologic hypoxia” regulate cellular and nutrient metabolism, maintains healthy barrier, thereby maintaining systemic homeostasis.

4. HIFs IN GUT & LIVER PATHOBIOLOGY:

While physiological hypoxia and HIFs play important roles in normal cellular functions, states of pathologic hypoxia/chronic HIF activation leads to tissue injury and dysfunction. This section will focus on two pathologies characterized by pathologic hypoxia, inflammatory bowel disease and non-alcoholic and alcoholic fatty liver disease.

4.1 Inflammatory bowel disease (IBD) is a heterogeneous, chronic inflammatory diseases of the gastrointestinal tract and comprises two major subtypes: ulcerative colitis and Crohn’s disease. Although the etiology of IBD is complex and not completely understood, an interaction of environmental (diet, lifestyle), genetic, immune, and gut microbial factors contribute to the pathogenesis. These factors ultimately converge to disrupt the homeostasis of intestinal epithelial cells, leading to compromised mucosal barrier function and persistent inflammation (57). IBD patients are prone to develop complications like fibrosis, anemia, fistulas, abdominal abscesses and have increased risk to develop colorectal cancer (CRC). The current class of IBD therapeutics is aimed at ameliorating intestinal inflammatory responses and maintaining clinical remission which include drugs like mercaptopurine, biologics (infliximab, ustekinumab, vedolizumab). New targets such as Janus Kinase, integrin inhibitors, sphingosine-1-phosphate receptor modulators are being tested in clinical trials with promising results (58). Nevertheless, despite tremendous advances being made in the last few decades, challenges remain in IBD care; (i) not all patients respond to therapies (ii) ~40% of initial responders lose response over time and fail to respond to other IBD therapies (iii) the clinical remission rates for therapies range from ~ 20–50% and majority of patients eventually require surgical interventions. (iv) unwanted drug side effects like immune suppression, high risk of infectious diseases and malignancy (58). As disruption of intestinal epithelial barrier is central in IBD pathogenesis, a deeper understanding of the mechanisms regulating intestinal barrier in homeostasis and injury may lead to targeted therapeutics. Fatty liver disease (FLD) is characterized by a broad histological spectrum spanning from accumulation of liver fat (or simply hepatic steatosis) to cirrhosis and is further categorized into non-alcoholic (NAFLD) or alcoholic (AFLD) fatty liver disease (59). While the underlying mechanisms of fatty liver disease are complex and multifactorial, dysregulation of hepatocyte lipid metabolism and ensuing chronic inflammatory responses are essential in disease progression (59). NAFLD and AFLD patients are at increased risk of developing nonalcoholic steatohepatitis (NASH) or alcoholic steatohepatitis (ASH) respectively and progression to end stage liver diseases such cirrhosis and hepatocellular carcinoma. There are no FDA approved drugs for FLD, and gradual weight loss is typically recommended to treat NAFLD or abstinence from alcohol and corticosteroids remain mainstay treatments of alcoholic hepatitis. Thus, a better understanding of the mechanisms contributing to fatty liver onset and progression may lead to new therapies.

4.2. Intestinal epithelial and hepatocyte HIFs in IBD and FLD:

As both IBD and FLD are characterized by pathologic hypoxia and HIFs participate in intestinal epithelial barrier and hepatic lipid metabolism and inflammatory response, studies have focused on understanding the role of HIFs in gut and liver pathobiology.

4.2a: Intestinal epithelial HIFs in IBD:

Hypoxia is a notable feature of the inflammatory foci in IBD, and increased expression of HIF1α and HIF2α has been observed in human specimens and preclinical IBD models (25; 60). As mentioned earlier, HIF1α regulate the expression of several proteins essential for maintaining intestinal barrier (18). Accordingly, genetic deficiency or stable expression of intestinal epithelial HIF1α was sufficient to exacerbate or ameliorate colitis respectively (60; 61). HIF1α activation was further found to modulate mTOR/NLRP3 pathway, improving inflammation in Crohn's disease (62). While HIF1α resolved IBD, HIF2α activation progressed disease. We reported that HIF2α activation compromised barrier integrity (51), deregulated mitochondrial iron homeostasis (63) and increased the expression of inflammatory mediators like macrophage migration inhibitory factor (64) and TNFα (25; 65) increasing colitis. While constitutive HIF2α overexpression in intestinal epithelium was observed to be lethal as mice succumbed to massive intestinal injury (25), temporal intestinal HIF2α activation in adult mice did not cause any basal intestinal injury, however, it was sufficient to increase inflammatory mediators like TNFα (65). With additional insults, temporal activation of intestinal HIF2α promoted colitis (65) Recently, TNFα was shown to promote intestinal mucosal repair (66). As large number of IBD patients receiving biologics like infliximab fail to respond, these findings suggests that neutralizing TNFα may interfere with colonic mucosal healing processes in these settings. Likewise, whether HIF2α/TNFα axis play a role in mucosal repair processes remains to be determined, it is noteworthy to speculate that HIF2α activation may be beneficial by facilitating repair processes in early-stage disease, however, in late-stage disease and with additional insults may aggravate IBD. As HIFs are regulated by PHDs and FIH enzymes, the role of PHDs (1–3 isoforms) have also been characterized in IBD. Genetic disruption of PHD1 (but not PHD2 and PHD3) protected in mouse models of colitis (67–69). Similarly, intestinal deletion of FIH was shown to reduce inflammatory response and colitis (61). Consistent with this data, several pan-PHD inhibitors were shown to improve colitis in animal models (70; 71) prompting initiation of clinical trials in IBD (72). In addition to cell autonomous roles, epithelial HIF1α exerted cell non-autonomous roles. Epithelial deletion of HIF1α was found to disturb intraepithelial lymphocytes homeostasis (73) and cause dysbiosis (74), increasing colitis. Collectively, intestinal epithelial HIFs have divergent roles with HIF1α being protective and HIF2α being injurious in colitis (Figure 2A).

Figure 2: HIF in chronic inflammatory disorders of the intestine and liver.

A) Intestinal HIFs in inflammatory bowel disease: Intestinal HIF1α activation regulate expression of mucins, integrins, CD73, tight junction proteins, maintain intraepithelial lymphocytes homeostasis and inhibits inflammation ameliorating colitis. Intestinal HIF2α activation increases turnover of tight junction proteins, deregulate mitochondrial iron homeostasis, and increases the expression of inflammatory mediators potentiating colitis. B) Hepatocyte HIFs in fatty liver disease: Chronic hepatocyte HIF1α and HIF2α activation deregulate liver lipid metabolism, decreases β- oxidation, potentiates inflammation and fibrosis increasing hepatic steatosis. Acute hepatocyte HIF2α inhibits glucagon improving glucose homeostasis. (C) Intersection of gut/liver axis in IBD and FLD: Chronic intestinal HIF2α activation increases ceramides and hepatic steatosis, while L-cells HIF2α activation increases GLP-1 protecting against steatosis. Intestinal epithelial loss of HIF1α exacerbate AFLD. Inhibition of HIF2α increased hepatic bile acid pools promoting intestinal regeneration and protect against colitis. Abbreviations: AFLD, alcoholic fatty liver disease; CXCL1, C-X-C motif chemokine ligand 1; Fe2C, iron; FLD, fatty liver disease; GLP-1, glucagon-like peptide-1; HIF, hypoxia-inducible factor; IBD, inflammatory bowel disease; MIF, migration inhibitory factor; PPARa, peroxisome proliferator activated receptor alpha; TNF, tumor necrosis factor.

4.2b: Hepatocyte HIFs in FLD:

HIFs are highly induced in NAFLD and AFLD. Mouse models using hepatocyte-specific deletion and overexpression of HIFs have shed light on the role of HIFs in FLD. While hepatocyte specific HIF2α overexpression deregulated lipid metabolism, increased lobular inflammation, parenchymal liver injury and fibrosis aggravating nonalcoholic fatty liver progression, deletion of hepatocyte HIF2α had an opposite effect suggesting that hepatocyte HIF2α is detrimental and drives fatty liver injury (40; 75; 76). Interestingly, conflicting evidence have emerged regarding the role of hepatocyte HIF1α in NAFLD and AFLD. Some reports have suggested a protective role (77) while others have indicated a detrimental role (39; 78). Furthermore, digoxin, a selective HIF1α inhibitor was shown to ameliorate inflammation and protect against ASH and NASH (79). Hepatocyte HIFs participate in glucose homeostasis (33; 34), suggesting that HIFs have complex relationship with glucose and lipid metabolic pathways, where their activation can have opposite effects dependent on the context and duration of activation (Figure 2B). Therefore, caution must be exercised when targeting HIFs in FLD. Chronic inhibition of HIF2α to relieve hepatic lipid accumulation may increase hepatic glucose production, thereby affecting systemic glucose metabolism and increase the risk of type 2 diabetes.

4.2c: Intersection of gut/liver axis in IBD and FLD:

The gut/liver axis play important roles in maintaining systemic metabolic homeostasis and communicate via biliary system and systemic circulatory mediators. Intestinal factors shape hepatic glucose and lipid metabolism and conversely, liver factors such as bile acids influence the gut microbial communities and barrier integrity (80). As HIFs regulate gut and liver physiology, crosstalk between gut and liver HIFs have also emerged as key players influencing gut and liver pathology. Xie et al reported a positive correlation between intestinal HIF2α expression and body mass index (BMI) and liver toxicity. The authors showed that activation of intestinal HIF2α during obesity increased ceramide synthesis fueling hepatic steatosis (28). However, Mooli et al reported that intestinal HIF2α regulated lipid sensing and increased GLP-1 secretion, thereby protecting against diet induced obesity and hepatic steatosis (81). These contradictory findings suggests a complex role of intestinal HIF2α influencing hepatic steatosis which may further be attributed to activation of HIF2α in specific intestinal cell type, enterocytes compared to enteroendocrine L-cells. Furthermore, intestinal dysbiosis aggravate NAFLD and AFLD and intestinal epithelial loss of HIF1 was reported to exacerbate AFLD (82) (Figure 2C). Liver is the primary organ responsible for bile acid synthesis from cholesterol. Once synthesized, primary bile acids cholic acid (CA), chenodeoxycholic acid (CDCA) are conjugated to glycine or taurine and transported to intestine via the biliary tract whereby they participate in wide variety of functions like emulsification and absorption of dietary cholesterol, fats, and fat-soluble vitamins, modulating intestinal microbial composition, intestinal barrier (83). ~ 95% of conjugated bile acids are re-absorbed in the terminal ileum while ~ 5% undergo chemical alterations like deconjugation by gut microbiota. Accumulating evidence suggests that IBD patients exhibit dysregulated bile acid metabolism whereby there is an increase in primary bile acids and subsequent decrease secondary bile acids correlating with disease severity (84). Rectal supplementation of secondary bile acids was shown to ameliorate experimental colitis via activation of TGR5 bile acid receptor (85). HIFs participate in bile acid regulation. Loss of intestinal HIF2α was reported to increase the abundance of bile acids conjugates, improving metabolic homeostasis (86) Likewise, activation of hepatic HIF2α inhibited bile acid synthesis (87) while its inhibition increased hepatic bile acid pools (88). Interestingly cholecystokinin supplementation to enhance the release hepatic bile acids in the intestinal lumen was shown to promote intestinal regeneration protecting against colitis (89) (Figure 2C). Although undetermined, it is plausible to speculate that increasing hepatic bile acid pools via HIF2α inhibition may protect against IBD. As discussed in 3.2, gut and liver regulate systemic iron homeostasis whereby intestinal iron absorption response is dependent on HIF2α. However, whether liver-derived hepcidin and intestinal HIF2α crosstalk to maintain iron homeostasis was not clear. In an elegant study, Schwartz et al showed that liver-derived hepcidin controlled intestinal specific HIF2α response by inhibiting iron dependent intestinal prolyl hydroxylase enzymes activity and thereby regulated iron uptake during systemic iron deficiency and iron overload conditions. Moreover, intestinal HIF2α inhibition reversed liver, heart and pancreatic iron accumulation in hepcidin deficient hemochromatosis model suggesting that inhibition of intestinal HIF2α may be a useful strategy for treating iron overload disorders (46). Altogether, intestinal and liver HIFs not only influence IBD and FLD independently but crosstalk between gut and liver HIFs influence gut and liver pathology.

4.3. Immune HIFs in IBD and FLD:

There is now considerable evidence to suggest that cellular metabolism alters immune cell function and differentiation with HIFs playing key roles in cellular metabolism (23). Innate and adaptive immune cells play important roles in IBD. Genetic modulation of HIFs in distinct immune cells has unveiled complex and contradictory roles of immune HIFs. A study reported that disruption of HIF1α in T cells lead to expansion of Th1, Th17 cells and increased colitis (90). However, another study showed that activation of HIF1α in T cells via silencing of PHDs increased IFNγ-secreting Th1 cells and promoted colitis (91). Tregs are indispensable for suppressing tissue inflammation. Loss of HIF2α in Tregs impaired Tregs function and increased colitis (92). In addition to T cells, innate immune HIFs participate in IBD, and studies have shown both similar and opposing roles myeloid HIF1α and HIF2α. While myeloid HIF1α was found to be indispensable for the glycolytic capacity and inflammatory mediators and loss of it resulted in decrease colitis (93), hematopoietic PHD1 deficiency protected against colitis via increased polarization of M2 macrophages (94). On the other hand, knockout of myeloid HIF2α aggravated colitis which was attributed to heightened inflammation by neutrophils (95) PHD3 was found to be essential for hypoxic neutrophil survival and loss of PHD3 reduced neutrophilic inflammation in a colitis model (96). Likewise, dendritic specific HIF1α null mice were susceptible to colitis via impaired Tregs (97) and PHD1 deficiency in dendritic cells significantly reduced inflammatory responses (94). Lastly, HIF1α induced CD11b expression in B cells was essential in regulating the immunosuppressive function of B cells and protected in IBD (98). Immune cells play important roles in liver injury. In an acetaminophen induced liver injury model (APAP), loss of T cell specific HIF1α was shown to inhibit γδ T cell infiltration and inflammatory response, thus suppressing injury (99). CD69+CD103-CD8+ T cells are a major population of human liver CD8+ T cells exerting intrahepatic immune responses. CD69+CD103-CD8+ T cells were found to be dependent on HIF2α as inhibition of HIF2α led to suppression of the effector functions. Consistent with this data, upregulation of HIF2α levels in CD69+CD103-CD8+ T correlated with liver failure in cirrhotic patients (100). Macrophage HIFs participate in FLD, whereby deletion of macrophage HIF1α abrogated inflammatory responses and decreased hepatic steatosis (101), liver fibrosis (102) and deletion of HIF2α exacerbated APAP (103). These results suggest a complex role of immune HIFs in the progression or resolution IBD and FLD. Thus, a better understanding of the nuances of immune HIFs and their contribution at different stages of the disease may provide specific targets for therapeutic intervention (Table 1).

Table 1:

Roles of HIF1α and HIF2α in IBD, FLD and CRC, HCC

	HIF1α (IBD and FLD)	HIF2α (IBD and FLD)
Intestinal Epithelium Hepatocyte	IBD: Regulate expression of barrier genes, antimicrobial peptides, mucins, decrease inflammatory mediators (48–50; 53–55) and protect in IBD (60–62;67;70–72) FLD: Regulate glucose metabolism (31;32) & lipid metabolism and promote (39,78;79) or protect (77) FLD	IBD: Chronic activation compromise barrier, increase inflammatory mediators and promote IBD (25;51;63–65) FLD: Regulate glucose metabolism (22;33;34;36) & lipid metabolism and promote FLD (40;75;76)
Macrophages	IBD: Indispensable for the glycolysis, promote inflammatory mediators & colitis (93) Loss of PHD1 polarized M2 MΦand protected in colitis (94) FLD: Promote inflammatory responses and hepatic steatosis and fibrosis (101;102)	IBD: Dampens neutrophil induced inflammatory response and protects in colitis (95). FLD: Protects against liver injury (103)
Dendritic	IBD: Promote Tregs function (97), loss of PHD1 suppressed inflammation (94)	IBD and FLD: Not characterized
Neutrophils	Loss of PHD3 reduced inflammation in colitis (96) and neutrophil survival (165)	Promote neutrophil survival (165) and motility (166)
T cells	IBD: Inhibit (90) or promote (91) expansion of Th1 cells and increased colitis FLD: Promote T cell infiltration, increase inflammatory response, aggravate liver injury (99)	IBD: Indispensable for Tregs function and protects in colitis (92) FLD: Critical for CD69+CD103-CD8+ T cells effector functions and promote liver cirrhosis (100)
B cells	IBD: Regulate the immunosuppressive function and protects in IBD (96)	IBD and FLD: Not characterized
Stromal	IBD: Anti-inflammatory properties, improves Crohn’s disease (105), colitis (106). FLD: Promote fibrosis (105)	FLD: Promote fibrosis (105)
	HIF1α (CRC and HCC)	HIF2α (CRC and HCC)
Intestinal Epithelium (CRC) Hepatocyte (HCC)	CRC: Does not alter CRC (115) HCC: Promote metastasis, angiogenesis, macropinocytosis, immune evasion responses driving HCC (123; 132; 133; 154–156)	CRC: Promote oncogenic signaling, deregulate iron metabolism, increase inflammatory responses, promote CRC (112;114;148) HCC: Promote oncogenic signaling, deregulate lipid metabolism, increase fibrosis and inflammatory mediators and promote (113; 116) or inhibit (129) HCC
Macrophage	CRC: Loss of PHD2 increase CRC (164). HCC: Promote Tregs infiltration, oncogenic signaling driving HCC (165;166)	CRC: promote inflammatory response and chemoresistance driving CRC (162;163) HCC: promote HCC (162)
T cells	promote Th17 development and CRC (159)	Indispensable for Tregs function and aggravate CRC (92)
Stromal	CRC: Regulate CAF TGF-β2 secretion driving CRC (173) HCC: Promote metastasis (177)	CRC: upregulated laminin alpha in endothelial cells, promoting immune responses and inhibit CRC (175) HCC: Not characterized

4.4. Stromal HIFs in IBD and FLD:

Intestinal stromal cells consists of heterogenous population of non-hematopoietic, non-epithelial, and non-endothelial cells and include mesenchymal stromal cells, fibroblasts, myofibroblasts, smooth muscle cells. Intestinal stromal cells are known to express HIFs and have regenerative and immunosuppressive properties (104). Although the role of HIFs in stromal cells is understudied, some data suggests that stromal HIFs could be exploited in IBD and FLD. MSCs stably expressing HIF1α were shown to exhibit anti-inflammatory properties and improved experimental Crohn’s disease (105). Moreover, hypoxic preconditioning of mesenchymal cells promoted the immunosuppressive effects and protected in colitis models (106). Liver fibrosis is a key pathological feature of progressive FLD, and hepatic stellate cell (HSC) activation drives liver fibrosis. Disruption of HIF1α inhibited HSC activation, whereas activation of HIF1α and HIF2α in HSCs was sufficient to increase pro-fibrotic mediators (107) (Table 1).

5. HIFs IN GUT & LIVER ONCOGENESIS:

Tumoral hypoxia is a hallmark of solid cancers including colorectal cancer (CRC) and hepatocellular carcinoma (HCC) and occurs due to combination of increased oxygen consumption by uncontrolled tumor cell proliferation, immune infiltration, and impaired blood supply due to defective formation of tumoral blood vessels. HIFs contribute to pro-tumorigenic cellular metabolism, proliferation, angiogenesis, metastasis, and drug resistance (41). This section will focus on the role of HIF signaling in colorectal cancer (CRC) and hepatocellular carcinoma (HCC)

5.1 Colorectal cancer (CRC) is the second leading cause of cancer deaths worldwide and involve a complex interplay of genetic and environmental risk factors (108). CRC occur sporadically in vast majority of cases with the initiating event being loss of tumor suppressor, Adenomatous polyposis coli (APC) and ~ 25% of patients have family history or genetic predisposition. Premalignant colonic polyps are believed to progress through adenoma-carcinoma model by acquiring progressive genetic alterations and mutations in tumor suppressors like APC, TP53, SMAD4 and oncogenes like EGFR, PIK3CA, KRAS, BRAF (109). Current treatment regimens include surgery and chemotherapy such as fluorouracil, leucovorin, oxaliplatin, and irinotecan. Although extensive screening has reduced incidence and mortality of CRC patients older than 50 years, survival is significantly decreased at advanced stages of metastatic disease. Interestingly, CRC incidences have risen in younger population mainly attributed to dietary and lifestyle changes. Thus, there is a pressing need to better understand the onset and progression of CRC biology to develop targeted therapies (108). Liver cancer is the fastest rising cause of cancer-related deaths worldwide, with the median life expectancy < 2 years (110) It is comprised of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) accounting for ~90% and ~5% respectively of all liver cancer cases. Chronic liver infections like hepatitis B, C virus, nonalcoholic and alcoholic steatohepatitis are the most prominent risk factors for HCC development which ultimately progress to fibrosis and cirrhosis, culminating in hepatocellular carcinoma. Current HCC treatments include surgery, liver resection and transplantation, radiotherapy, chemotherapy like tyrosine kinase inhibitors (viz. sorafenib, lenvatinib, cabozantinib) and immune checkpoint inhibitors like Nivolumab (111). Although our understanding of the mechanisms of CRC and HCC progression has significantly improved over the last few decades, current treatment options provide modest survival benefits, and often encounter drug toxicities and resistance. CRC and HCC are characterized by intra-tumoral hypoxia, and patient data suggest that HIF1α and HIF2α are prognostics indicators of disease severity in CRC (112) and HCC (113). In line with these observations, animal studies have shown that HIFs participate in CRC, (112; 114; 115) and HCC (113; 116) . However, there remain gaps in our knowledge about the role of tumoral cell intrinsic HIF signaling and its crosstalk between different tumoral cell types in tumor microenvironment driving CRC and HCC. Identifying such mechanisms will further our understanding and may provide new targeted therapeutic opportunities in CRC and HCC.

5.2. HIFs in oncogenic signaling in CRC and HCC:

Hyperactivation of Wnt/β-catenin signaling is the initiating event in CRC and a critical pathway for HCC. HIFs have divergent roles in modulating Wnt/β-catenin signaling. HIF2α regulate β-catenin activity by direct and indirect mechanisms. HIF2α was shown to directly interact with β-catenin (117), or increase WNT5A expression (118) thereby enhancing β-catenin/TCF-driven transcriptional responses. However, HIF1α negatively regulated Wnt/β-catenin signaling (119). In CRC, oncogenic signaling pathways, including EGFR, IGF1R, PIK3CA, and downstream effectors, are driven by HIF2α, which acts as a central node of convergence (120). In HCC, HIFs were also shown to modulate Wnt/β-catenin, PI3K/Akt, hedgehog and Hippo/YAP to drive tumor growth (116; 121). Oncogenic signaling alter cellular biosynthetic pathways and deregulate nutrient metabolism to support growth. The tumor microenvironment is nutrient stressed and recent studies suggest that tumors deploy macropinocytosis to scavenge extracellular protein as a nutrient source (122) HIF1α was shown to be a key facilitator of macropinocytosis driving HCC (123). HIF1α was further shown to increase proliferation via regulation of Forkhead box M1 (124), cyclin A, D & decrease apoptosis via induction of Bcl2 (125). Moreover, deletion of HIF1α disrupted glycolytic or lipid metabolism limiting HCC (126). Recently, disruption of hepatocyte-specific HIF2α was found to decrease NASH associated HCC via reduced Hippo/YAP signaling (113). Further, silencing of HIF2α synergized with doxorubicin (127) or sorafenib (128) to suppress HCC. However, one study found that enforced HIF2α expression increased apoptosis and inhibited HCC (129). HIFs regulate angiogenesis and a well characterized HIF angiogenic target is vascular endothelial growth factor (VEGF). Expression of HIFs directly correlate with VEGF expression and micro-vessel density and silencing of HIF2α led to significant decrease in VEGF (130). In a phase II study, fluorouracil, leucovorin, oxaliplatin, and irinotecan (FOLFOXIRI) plus angiogenic inhibitor bevacizumab improved the outcome in patients with metastatic CRC (131). Sorafenib was found to reduce HIF1α, VEGF, thereby limiting HCC vascularization and growth (132). In addition to promoting angiogenesis, HIF1α is a critical regulator of the epithelial-mesenchymal transition (EMT) which is essential for invasion and metastasis. HIF1α regulates the expression of several key genes including snail homolog 1, E-cadherin, N-cadherin, and vimentin. Silencing of HIF1α significantly suppresses the migration and invasion of CRC and HCC cells (132; 133).

5.3. HIFs in iron metabolism in CRC and HCC:

Oncogenic signaling rewires nutrient metabolism (glucose, amino acids, lipids) to achieve uncontrolled growth. Additionally, cancer cells require huge quantities of micronutrients like minerals and vitamins to support these biological activities and one such vital micronutrient is iron (134). Dysregulated iron metabolism is a key feature of CRC and HCC, whereby tumors accumulate large quantities of iron. Immunostaining of colon and liver tumor biopsies showed significant increase in iron import and decrease in iron export compared to normal tissues (135; 136) suggesting that intra-tumoral iron content may be important to drive CRC and HCC. Indeed, feeding mice a low iron diet significantly decreased tumor proliferation and growth in sporadic and colitis – associated CRC and NASH driven HCC models (112; 136). These observations also extended to human subjects whereby patients undergoing phlebotomy were found to have lower risk and mortality rate of CRC (137) and HCC (138). Moreover, hereditary hemochromatosis increases the risk for HCC and iron chelators showed positive outcome in a small cohort of advanced HCC patients (139). As HIF2α is intimately linked with intestinal iron homeostasis, HIF2α induced iron metabolism have been studied in CRC. Intestinal iron importer, DMT-1 was found to be essential for HIF2α induced colon tumor growth and genetic deficiency, or pharmacological inhibition of DMT1 reduced colon tumors (114). Iron have been reported to activate oncogenic signaling cascades such as Wnt/β-catenin, signal transducer and activator of transcription 3 (STAT3) and increase reactive oxygen species (140). In support of this, HIF2α induced iron import was found to be essential in activating CDK1/JAK/STAT3 pathway and blunting DMT1 decreased STAT3 activity and CRC growth(114). As STAT3 is one of the major oncogenic pathways activated in CRC, these findings suggests that HIF2α is central in deregulating tumor iron homeostasis and fueling oncogenic signaling in CRC. Interestingly, while DMT1 significantly contributed to CRC iron accumulation, DMT1 was found to be dispensable for hepatic iron accumulation (141). Contrarily, HCC iron accumulation was attributed to increased transferrin receptor (TFRC) which further correlated with increased vascular invasion and poor survival outcomes (142). As HIF2α regulate hepatic TRFC (143), these findings suggests that HIF2α may contribute to HCC iron accumulation. Apart from iron import genes, HCC and CRC patients’ biopsies also exhibit reduced expression of ferroportin, the only known cellular iron exporter (135; 136). Loss of colonic epithelial ferroportin was shown to increase growth (135) and reduced ferroportin expression was associated with higher lymph node staging in HCC (142). As hepcidin is the only known regulator of ferroportin, the question arose whether colon tumors locally expressed hepcidin to degrade ferroportin and modulate tumoral iron. CRC tumors were shown to express hepcidin which was partly driven by HIF2α and loss of colonic epithelial hepcidin decreased tumors in a CRC sporadic model (135). Surprisingly, HCC tumors significantly downregulated hepcidin suggesting that ferroportin downregulation occurs via other mechanisms (144). Furthermore, a positive correlation of hepcidin was associated with increased tumor infiltration of cytotoxic T cells suggesting that liver tumors may actively downregulate hepcidin promoting immunosuppressive microenvironment independent of iron handling (144). Further studies are warranted to understand the role of hepcidin in HCC. Excessive tumoral iron can be toxic due to increased generation of reactive oxygen species via the Fenton reaction and thereby can act as a double edge sword. Identifying iron dependent tumor specific vulnerabilities can lead to new targets. In this regard, Ferroptosis, a nonapoptotic, iron-dependent form of oxidative cell death characterized by lipid peroxidation has emerged as a potential target for CRC and HCC (145). HIF2α was reported to increase ferroptosis resulting in CRC lethality (146). Sorafenib, the first-line therapy for advanced HCC has been found to induce ferroptosis. However, ferroptosis alone did not suppress HCC development, but instead, stimulated the recruitment of immunosuppressive MDSCs, resulting in a favorable immunosuppressive environment. In line with these findings, combination of the ferroptosis inducer with immune checkpoint blockade resulted in HCC lethality (147). These findings suggest that immune response induced by ferroptosis could be exploited as therapeutic target in HCC. It is unclear whether HIF2α contributes to HCC ferroptosis and lethality. Moreover, it is also unclear how tumor cells buffer excessive iron in addition to channeling it to fuel oncogenic signaling and nucleotide synthesis. Identifying such mechanisms may further our understanding of the mechanisms driving CRC and HCC. Collectively, CRC and HCC tumor cells use diverse mechanisms to deregulate tumor iron metabolism, fueling myriad oncogenic signaling pathways, with HIFs playing pivotal roles (Figure 3A and 3B).

Figure 3: HIFs in colorectal cancer and hepatocellular carcinoma.

A) Colonic tumoral HIF2α increases DMT1 to fuel Fe²⁺ influx and regulate hepcidin/FPN axis to inhibit Fe²⁺ efflux thereby potentiating oncogenic signaling. HIF2α increases inflammatory mediators increasing CRC. B) Hepatocyte HIFs promote macropinocytosis, dysregulate lipid metabolism, drives tumoral inflammation and immune evasion driving HCC. Abbreviations: CRC, colorectal cancer; CXCL1, C-X-C motif chemokine ligand 1; DMT1, divalent metal transporter 1; Fe2C, iron; FPN, ferroportin; HCC, hepatocellular carcinoma; HIF, hypoxia-inducible factor; HRGP, histidine-rich glycoprotein; IL, interleukin; MIF, migration inhibitory factor; ROS, reactive oxygen species; TfR1, transferrin receptor 1; TNF, tumor necrosis factor.

5.4. HIFs in inflammatory signaling in CRC and HCC:

Tumor promoting chronic inflammation is a hallmark of cancer (41). IBD patients are at increased risk of developing CRC in their lifetimes (57). Similarly, HCC is driven by inflammation and major risk factors are hepatitis B and C infections and alcoholic and non-alcoholic FLD (113). Hypoxia play a key role in mediating inflammatory responses, thereby shaping the tumor microenvironment, and driving tumor progression. Tumor hypoxia modulates inflammatory signaling and conversely, tumor inflammation potentiates HIFs signaling. In mouse models, HIFs have both cell autonomous and non-autonomous inflammatory roles in CRC and HCC. For instance, epithelial HIF2α was shown to drive CRC by activating COX2/PGE2 axis (148) while hepatocyte HIF2α aggravated lobular inflammation (149). Nuclear Factor-Kappa B (NF-κB) is a key transcriptional factor regulating HIFs and inflammation. Accordingly, there was a direct association between NF-κB, HIF1α, VEGF expression and metastasis in stage III human CRC (150). As HIFs alter the expression of cytokines, chemokines, and their receptors, these not only mediate tumor elicited inflammation but also results in innate and adaptive immune cells infiltration and ensuing inflammation. Epithelial HIF2α was found to increase polymorphonuclear neutrophils (PMNs) recruitment and colon tumors in CXCL1/CXCR2 dependent manner (151). Tumoral HIF1α was shown to recruit monocytes/macrophages and alter their polarization to immunosuppressive subtype via lactic acid (152). Likewise, HIF1α/IL-1β signaling between HCC cells and tumor-associated macrophages aggravated inflammatory microenvironment driving HCC metastasis (153) HIF1α regulates CD73 expression and thereby affect adenosine availability (49) As Adenosine signaling has emerged as a key immunosuppressive metabolic pathway and HIF signaling participates in drug resistance, these findings suggests that HIFs may dictate responses to cancer immunotherapies. In line with this, HIF1α was shown to reprogram adenosine metabolism promoting HCC (154) . Furthermore, inhibition of HIFs in HCC was shown to reprogram the tumor immune microenvironment, improving immune therapy responses (155) and HIF1α was essential to maintain myeloid-derived suppressor cells promoting immune evasion in HCC (156). Collectively, intestinal epithelial HIFs drive CRC and HCC via (i) promoting cell autonomous inflammation (ii) recruiting innate and adaptive immune cells resulting in heightened inflammatory and immune suppressive milieu.

5.5. Immune HIFs in CRC and HCC:

Hypoxic tumor microenvironment is known to confer resistance to chemo-, radio- and immune-therapies (121). As immune cells are exposed to a hypoxic tumoral microenvironment, immune cell intrinsic HIFs play important roles in CRC and HCC biology and could either promote or suppress tumor progression. Tumor-infiltrating CD8 cytotoxic lymphocytes are majorly responsible for tumor killing and are targets of immunotherapies, aimed to relieve the brake of T cell suppression (157) Although T cell receptor stimulation increases HIF1α/HIF2α expression, HIFs were reported to have divergent roles in CD4 and CD8 effector function. While depletion of CD4 HIF1α significantly enhanced inflammatory responses, depletion of CD8 HIFs increased effector responses resulting in decreased tumor growth (158). Tregs play a vital role in dampening CD8 responses and promote immune suppressive tumor environment. Deletion of HIF2α in Tregs dampened their suppressive function resulting in decreased CRC (92). Th17 are critical inflammatory mediators in colitis associated cancer (CAC) and neutralizing IL17 dampened CAC (159) HIF1α were shown to regulate the retinoic acid–related orphan receptor-γt (RORγt) and Th17 differentiation (160). Thus, a balance of HIFs activation in CD4/CD8 cells may dictate tumor responses. Tumor associated macrophages (TAMs) and tumor associated neutrophils (TANs) can also have pro- and anti-tumor functions (161). Macrophages could be simply classified as M1 (anti-tumor) or M2 (pro-tumor) and are known to play roles in CRC and HCC. Macrophage HIF2α was shown to regulate inflammatory cytokine expression and deletion of macrophage HIF2α decreased tumor burden in CAC model (162). TAMs were shown to express dihydro-pyridine dehydrogenase in a HIF2α dependent manner leading to inactivation of 5FU and chemoresistance to 5-FU in CRC (163). PHD2 deficiency in hematopoietic cells increased CAC growth via increased the number of tumor-associated macrophages and increased oncogenic signaling (164). In HCC, HIF2α increased TAM infiltration (162), while HIF1α increased TREM-1 expression, Tregs infiltration (165) driving HCC. Macrophage HIF1α was reported to increase oncogenic signaling driving HCC in diet-induced obesity and diabetes models (166). HIF1α and HIF2α was shown to promote neutrophil survival (167) and HIF2α was shown to be direct regulator of neutrophil motility (168). HIFs polarize TANs to an immunosuppressive phenotype via increased TGF-β to promote HCC (169). In addition to fostering immunosuppressive environment, TAMs and TANs promote angiogenesis. Macrophage and neutrophil HIFs were shown to induce VEGF and MMPs expression driving drug resistance (170). As VEGF impact the function of immune cells, these findings suggests that HIF/VEGF axis may not only affect tumor angiogenesis but also reprogram immune cells driving tumorigenesis. Interestingly, combination of an immune checkpoint inhibitor (atezolizumab) with anti-VEGF (bevacizumab) received FDA approval after reporting of increased overall and progression-free survival outcomes (171). MDSCs suppress the anti-tumor immunity and are modulated by HIFs. HIF1α was shown to activate PD-L1 in MDSCs, differentiate MDSCs into TAMs (172) with myeloid PD-L1 expression correlating with poor HCC survival. Thus, spatial distribution of hypoxic regions within the tumor microenvironment may dictate HIFs activation and the tumoral immune responses (Table 1).

5.6. Stromal HIFs in CRC and HCC:

There is now considerable evidence to suggest that stromal cells drive tumor proliferation, angiogenesis, and metastasis via secretion of growth factors and cytokines. Tumoral stromal cells include cancer associated fibroblast (CAFs), endothelial cells, pericytes, fibrocytes. As HIFs regulate proliferation, angiogenesis, metastasis, HIFs impact stromal cell biology. TGFβ is a potent immunosuppressive ligand secreted by CAFs and was shown to be dependent on HIF1α (173). Not surprisingly, genetic deletion ablation of this HIF1α/TGF-β2 axis reduced CRC Recently, CAF-specific HIF2α, but not HIF1α, promoted pancreatic tumor progression by fostering immunosuppressive microenvironment (174). Whether CAF HIF2α fosters an immunosuppressive environment in CRC remains to be determined. Lastly, enforced expression of endothelial superoxide dismutase (SOD3) upregulated laminin alpha 4 (LAMA4) via HIF2α and increased cytotoxic CD8 tumor infiltration, inhibiting CRC (175). Like CRC, CAF participate in HCC progression and hypoxia was shown to increase fibroblast activation protein (FAP), a CAF marker in HCC patients (176). Consistently, CAF-derived CCL5 promoted HCC metastasis via HIF1α/ZEB1 axis (177). Thus, future studies directed at deciphering the mechanisms by which stromal HIFs impacts CRC, HCC biology and how stromal HIFs crosstalk with other cells influencing the tumoral microenvironment will be vital (Table 1).

6. CONCLUDING REMARKS AND FUTURE PERSPECTIVES:

Significant progress has been made in understanding the complex microenvironment of gut and liver diseases, such as IBD, FLD, CRC, and HCC. Hypoxia is a hallmark of these diseases and affect disease biology. The role of hypoxia in the microenvironment is complex and integrated with dynamic elements that shape disease progression, immune response, and therapeutic resistance. The interactions between epithelial/parenchymal cell- autonomous and non-autonomous mechanisms shape the microenvironment and disease progression. Although cutting-edge technologies present a chance to investigate the basic biology of hypoxia in liver and gut diseases, there are still considerable gaps in our understanding, including insufficiently studied microenvironmental components and the intercellular relationships that hypoxia shapes. Recent research has focused on the role of hypoxia-induced signaling in gut and liver pathobiology. Several PHD inhibitors have been effective in preclinical animal models of IBD, but a phase II (NCT04556383) trial testing PHD inhibitors was terminated due to a lack of treatment benefit, suggesting that pan-PHD inhibitors may not be useful in IBD. HIF1α and HIF2α have opposing effects in IBD, and as pan-PHD inhibitors activate both HIF1α and HIF2α, the positive effects of HIF1α may be negated by HIF2α. Thus, selective activators of HIF1α, such as PHD1 selective inhibition, may be more effective as IBD treatments. Additionally, isoform specific PHD inhibitors should be considered due to the requirement of PHD3 for intestinal homeostasis (69). HIF2α plays a crucial role in driving CRC and HCC, and HIF isoform-selective inhibitors have been characterized. HIF2α selective blocker, belzutifan has been FDA approved for treatment of patients with Von Hippel-Lindau (VHL) associated cancers like renal cell carcinoma. Thus, HIF2α selective blockers could be repurposed and tested in iron overload disorders, IBD, CRC and HCC. Research advances described herein provide future investigations to inform novel strategies for improving HIF-based treatments.

OPEN QUESTIONS

• Understanding how HIF1α and HIF2α selectively drive gene expression may offer additional avenues to manipulate HIF signaling for therapy.

• Currently, there are viable therapeutics that can selectively activate or inhibit HIF1α and HIF2α, but their efficacy has not been extensively assessed in liver and gut pathologies. Additionally, combining selective modulators may enable an optimal HIF1α and HIF2α expression profile for treatment.

• New data is emerging with single-cell RNA seq that address heterogeneity of different cell types in tumor and inflammatory areas and together with assessing tissue pO2 and HIF expression may provide new insights into competitive and cooperative interactions of hypoxia in different cells of the microenvironment and allow a better of understanding the crosstalk between different cell types for precision treatment.

• Our understanding of hypoxic adaptation has mainly focused on HIFs. However, a complete picture of cellular hypoxic response remains unknown. Jain et al. attempted to address this question by conducting genetic CRISPR screens and identified hundreds of genes important for cellular fitness that were sensitive to O₂ fluctuations but independent of HIFs (178). Therefore, further investigation is needed to determine whether these hypoxia-dependent but HIF-independent genes participate in gut and liver pathology.

KEY HIGHLIGHTS.

• Physiologic oxygen gradients and HIF signaling in the intestinal epithelium and hepatocytes play a crucial role in maintaining the barrier function and liver zonation, respectively, thereby contributing to intestinal and liver homeostasis.

• Intestinal epithelial HIF1α provides protection in inflammatory bowel disease (IBD) and boosting HIF1α selective responses could be a strategic approach in IBD.

• Intestinal epithelial HIF2α disrupts nutrient metabolism, fuels oncogenic signaling driving colorectal cancer.

• Hepatocyte HIF2α activation promotes inflammatory responses and steatosis.

• Hepatocyte HIFs promote oncogenic signaling, deregulate iron metabolism and inflammatory responses in hepatocellular carcinoma.

• Tissue-resident and infiltrating immune cells experience changes in oxygen tension, and alterations in HIF expression in a cell-type-specific manner impact their function.