several studies , including our own , have demonstrated that regions of hypoxia develop in the liver after acute liver injury . early studies showed that hypoxia develops in the liver after alcohol treatment.12 , 13 later studies showed that damage to the liver with compounds such as monocrotaline or acetaminophen also produce regions of hypoxia.14 , 15 the mechanism by which this occurs is not known , but it most likely results from disruption of the hepatic architecture , which impedes blood flow through damaged regions ; activation of the coagulation system , which leads to fibrin clot formation in the vasculature ; and possibly production of vasoactive mediators that modulate hepatic blood flow to regions of injury . to identify regions of hypoxia , in addition to this method , activation of hif-1 has been used as a surrogate marker of hypoxia . hif-1 has been detected in the livers of mice treated with ethanol , acetaminophen , or carbon tetrachloride ( ccl4).13 , 18 , 19 , 20 in addition to hypoxia , however , hif-1 can be activated in cells by other mediators , including cytokines , growth factors , and oxidative stress . in fact , hif-1 is activated in the liver after acetaminophen and ccl4 treatment before the development of hypoxia , suggesting an important role for other mechanisms of hif-1 regulation in the liver after exposure to these toxicants.18 , 20 similar to acute injury , studies have shown that hypoxia is present in the liver during chronic injury . rosmorduc et  al21 , 22 originally showed hypoxia in the liver after chronic treatment of rats with diethynitrosamine or after bile duct ligation ( bdl ) , both of which produce severe fibrosis . we later confirmed these findings and demonstrated that hif-1 is activated in several cell types in the liver after bdl . in particular , hif-1 was activated in macrophages and hepatocytes within and at the periphery of regions of necrosis , both areas where hypoxia was present . in addition to animal models , we detected hif-1 protein in hepatocytes and scar - associated macrophages near regions of bridging fibrosis in livers from patients with primary biliary cirrhosis or primary sclerosing cholangitis . we also detected hif-1 in -smooth muscle actin ( -sma ) expressing myofibroblasts within regions of bridging fibrosis . because hif-1 regulates a number of genes that have been implicated in fibrosis development , including , platelet - derived growth factor ( pdgf ) , fibroblast growth factor-2 ( fgf-2 ) , vascular endothelial growth factor ( vegf ) , plasminogen activator inhibitor-1 ( pai-1 ) , and many others , our laboratory and others have conducted studies to evaluate the role of hif-1 in the development of fibrosis . hif-1 knockout mice die during embryonic development . accordingly , to test the hypothesis that hif-1 contributes to the development of liver fibrosis , our laboratory used cre - lox technology to knockout hif-1 in adult mice . in this study , hif-1 floxed mice were crossed with mice that express cre recombinase under control of the mx interferon - inducible promoter . in these mice , these mice and control mice that did not receive polyinosinic - polycytidylic acid were subjected to bdl . in hif-1-deficient mice , liver fibrosis was substantially reduced , which demonstrated for the first time a key role for hif-1 in the development of liver fibrosis in  vivo . deletion of hif-1 prevented up - regulation of several key profibrotic mediators including pdgf - a , pdgf - b , pai-1 , and fgf-2 . all these proteins have been implicated in the development of liver fibrosis , suggesting that hif-1 may promote fibrosis by regulating expression of these genes.30 , 31 , 32 , 33 in fact , studies have shown that these genes are directly regulated by hif-1 in some cell types.24 , 25 , 27 although this study indicated a key role for hif-1 in the development of liver fibrosis , the cell - specific role of hif-1 in the development of this disease remained unknown . as discussed earlier , hif-1 is activated in hepatocytes in mice subjected to bdl and in patients with primary biliary cirrhosis and primary sclerosing cholangitis.5 , 23 early studies by kietzmann et  al demonstrated that hif-1 is activated in hypoxic hepatocytes and that hif-1 directly regulates pai-1 in these cells . our laboratory confirmed these findings and also showed that hif-2 was activated in hypoxic hepatocytes and was needed for full induction of pai-1 in these cells . in addition to pai-1 , our studies demonstrated that hypoxia up - regulates vegf , adrenomedullin-1 ( adm-1 ) , and adm-2 in a hif-1 and hif-2-dependent manner . interestingly , although hif-1 regulates pdgf - a and pdgf - b in some cell types , these were not increased in hypoxic hepatocytes . our studies also showed for the first time an interaction between the hif and transforming growth factor  ( tgf- ) signaling pathways . in this study , our laboratory demonstrated that hypoxic hepatocytes activate latent tgf-1 in a hif - dependent manner . although the mechanism by which this occurs is not fully understood , we have evidence that hypoxia increases expression of several matrix metalloproteinases and thrombospondin-1 in hepatocytes ( copple , unpublished observations ) , all of which can activate latent tgf-1.36 , 37 , 38 collectively , these in  vitro studies demonstrated that hif-1 and hif-2 are activated in hypoxic hepatocytes and regulate expression of pai-1 and vegf as well as regulate activation of latent tgf-1 , which could promote the development of liver fibrosis ( figure  1 ) . since these studies , other laboratories have investigated the role of hepatocyte hifs in the development of liver fibrosis in  vivo . in a study by scott et  al , hif-1 ( aryl hydrocarbon nuclear transporter ) floxed mice were crossed with mice that express cre recombinase under control of the albumin promoter to generate hepatocyte - specific hif-1 knockout mice . as discussed earlier , hif-1 heterodimerizes with the basic helix - loop - helix - per - arnt - sim ( bhlh / pas ) transcription factor family , which includes hifs , and is thus required for both hif-1 and hif-2 transcriptional activity.6 , 9 mice with and without deletion of hif-1 in hepatocytes were treated with the hepatotoxicant thioacetamide to induce liver fibrosis . although the pattern of fibrosis was similar in both , there was a decrease in macrophage infiltration as well as a decrease in the mrna expression of profibrotic genes , including tgf-1 and tgf-2 , collagen type 1 and 5 , and tissue inhibitor of metalloproteinases 1 and 5 ( timp1 and  5 ) in hepatocyte - specific hif-1 knockout mice . these results provided support for the importance of hifs in regulating profibrotic gene expression in hepatocytes in  vivo after liver injury . further studies conducted by roychowdhury et  al also suggested an important role for hepatocyte hif-1 in the development of fibrosis . in their study , hepatocyte - specific hif-1deficient mice were fed ethanol concomitantly with ccl4 treatment , resulting in the amplification of ccl4-induced hepatic fibrosis . hepatocyte - specific hif-1deficiency prevented the ethanol - induced increase in ccl4-induced fibrosis compared with control mice , as measured by mrna and protein levels of both type i collagen and -sma , indicating an important role for hepatocyte hif-1 in the development of liver fibrosis in this model . however , when mice were treated with ccl4 alone without ethanol , hif-1 deficiency in hepatocytes did not reduce liver fibrosis , which may indicate that hypoxia resulting from ethanol metabolism may enhance hif-1 activation in hepatocytes after ccl4 treatment , allowing hif-1 to play a greater role in the development of liver fibrosis . studies by qu et  al found that hif-2 in hepatocytes may also be important for progression of fibrosis . in these studies , hepatocyte - specific vhl knockout mice were produced , which results in constitutively activate hif-1 and hif-2 in hepatocytes . gene expression profiles were then assessed in the livers of control mice and hepatocyte - specific vhl knockout mice . numerous genes critical to the development of liver fibrosis were found to be up - regulated . these included genes important for collagen formation , such as procollagen - lysine 2,oxoglutarate 5-dioxygenase 2 ( plod2 ) and transglutaminase 2 ( tgm2 ) as well as -sma . furthermore , vhl - disrupted mice fed an ethanol diet produced an increase in fibrosis . this increase was completely prevented in hepatocyte - specific vhl - hif-2 double knockout mice , but not in hepatocyte - specific vhl - hif-1 double knockout mice . together , these data indicated that hif-2 activation in hepatocytes is important for the development of liver fibrosis in this model . one caveat of this study , however , was that although deletion of hif-2 did prevent an increase in fibrosis , it also resulted in reduced liver injury overall . because liver injury is the driving factor leading to fibrosis , it is possible that hif-2 did not play a direct role in the progression of fibrosis , but rather played a role in producing liver injury . further studies are needed to more definitively elucidate the role of hif-2 in hepatocytes in the development of fibrosis . although these cells perform key beneficial functions , they also contribute to the pathogenesis of a number of liver disorders , including ischemia - reperfusion injury , toxin - induced liver injury , fatty liver disease , and liver fibrosis.43 , 44 rivera et  al were the first to show a key role for macrophages in the development of liver fibrosis in rats after chronic ccl4 treatment . subsequent studies have confirmed these results , reaffirming an essential role for these cells in the development of liver fibrosis . work from karlmark et  al indicated that the macrophage population that promoted fibrosis was recruited from bone marrow in a manner dependent on c - c chemokine receptor type 2 ( ccr2 ) and type 6 ( ccr6 ) . although these studies have clearly indicated that macrophages are important for the development of fibrosis , the mechanism by which they contribute to this process was not known . early studies by friedman and arthur showed that conditioned medium from cultured kupffer cells stimulated hsc proliferation in culture in a pdgf - dependent manner . in addition , studies provided evidence that intrahepatic cd11bf4/80gr1 macrophages could stimulate hscs to produce collagen in a tgf--dependent manner . consistent with these studies , immunohistochemical studies and in situ hybridization techniques have shown that macrophages express tgf- and pdgfs in the liver during the genesis of fibrosis.47 , 48 collectively , these studies demonstrated that macrophages are important for the development of fibrosis and that they may contribute to fibrosis by producing growth factors that stimulate hsc proliferation and collagen production . however , the mechanism by which chronic liver injury stimulated these cells to produce profibrotic mediators remained unknown . as discussed earlier , our studies demonstrated that hif-1 was activated in scar - associated macrophages in both mice  and humans with liver fibrosis.5 , 23 in subsequent studies , we showed that exposure of kupffer cells to hypoxia in  vitro activated hif-1 but not hif-2 , suggesting that hif-1 signaling may predominate in kupffer cells from nondiseased livers . this , however , could potentially change in diseased livers . in the presence of certain cytokines , macrophages polarize to different phenotypes , including classically activated ( m1 ) and alternatively activated ( m2 ) macrophages . takeda et  al showed that hif-1 is up - regulated and hif-2 is down - regulated in classically activated macrophages , whereas hif-2 is up - regulated and hif-1 down - regulated in alternatively activated macrophages . it is possible that kupffer cells in nondiseased livers are skewed toward a classically activated phenotype , which may explain the lack of hif-2 activation in these cells . in diseased livers , however , we have observed hepatic macrophages with activated hif-1 or hif-2 , indicating that both transcription factors can be activated in hepatic macrophages in  vivo after injury ( copple , mochizuki , and roth , unpublished observation ) . because hif-1 and hif-2 regulate overlapping and distinct sets of genes , it would be interesting to evaluate the pattern of gene expression in hypoxic kupffer cells either skewed toward a classic phenotype with activated hif-1 or an alternative phenotype with activated hif-2. it is possible that these two macrophage populations may play very different roles in liver disease in part due to differential expression of hif-1 and hif-2. studies have shown that hypoxic kupffer cells produce profibrotic mediators in  vitro . our laboratory showed that in  vitro exposure of kupffer cells to hypoxia increased expression of several genes involved in angiogenesis , including vegf and angiopoietin-1 , in a hif - dependent manner . in addition , pdgf - b was also up - regulated in hypoxic kupffer cells in a hif - dependent manner . in mice subjected to bdl , knocking out hif-1 selectively in macrophages using cre / lox did not affect liver injury , markers of cholestasis , or hepatic inflammation , including up - regulation of cytokines and hepatic neutrophil and macrophage accumulation . deletion of hif-1 did , however , reduce liver fibrosis as measured by type i collagen and -sma mrna and protein levels . in addition , knocking out hif-1 in macrophages prevented an increase in pdgf - b mrna and protein . it also prevented up - regulation of fgf-2 , another profibrotic growth factor , but did not affect levels of active tgf- protein . similar results were observed in mice in which hif-1 was knocked out in macrophages , suggesting that hif-1 in macrophages is profibrotic ( figure  1 ) . this is consistent with reports suggesting that classically activated , inflammatory macrophages primarily promote fibrosis . as mentioned earlier , hif-1 is up - regulated in classically activated macrophages . it would be interesting , however , to investigate the role of hif-2 in macrophages and determine whether it may play an opposing role and promotes fibrosis reversal . despite being originally described in the 19th century , it was not until 1985 that friedman et  al discovered that hscs are the main collagen - producing cell type in the liver . recent studies using sophisticated cell - tracing techniques have further confirmed the importance of these cells in collagen deposition during fibrosis . during quiescence , these cells store and release retinoids . after liver injury , these cells become activated and differentiate into -sma  expressing myofibroblasts that produce collagen , proliferate , and migrate throughout the liver . because liver injury produces regions of hypoxia , corpechot et  al tested the hypothesis that hypoxia stimulates hscs to produce collagen . in this study , exposure of rat hscs to hypoxia in  vitro up - regulated type i collagen , indicating that hypoxia is a stimulus for collagen production by these cell types . in addition , we recently confirmed these findings in culture - activated , primary human hscs ( copple , unpublished findings ) . although this process is not fully understood , it most likely evolved to provide an initial structural matrix to facilitate repair processes , such as angiogenesis . during chronic injury , however , persistent hypoxia provides a continuous stimulus for collagen production by hscs , leading to fibrosis . a later study by shi et  al confirmed that hypoxia up - regulates collagen in a human hsc line , lx-2 cells . in this study , they also observed an increase in tgf- , suggesting that hypoxia stimulates autocrine production of tgf- , which could stimulate collagen production . this same group also observed an increase in matrix metalloproteinase 2 , which could process tgf- to its active form . whether these processes require hifs and occur in  vivo during fibrosis development , however , remain to be determined . because of the results described here , our laboratory next determined whether hifs are activated in hypoxic hscs . in these studies , exposure of culture - activated , primary mouse hscs to hypoxia activated both hif-1 and hif-2. hypoxia also increased the expression of several genes that could contribute to fibrosis development . for instance , hypoxia increased expression of two genes involved in collagen metabolism , prolyl-4-hypdroxylase 1 and prolyl-4-hydroxylase 2 , which are key enzymes that contribute to the formation of stable collagen triple helices . as expected , hypoxia also increased expression of several genes involved in angiogenesis , including vegf , angiopoietin - like-4 , placental growth factor , and macrophage - migration inhibitory factor . it has been proposed that angiogenesis is a critical factor in the development of liver fibrosis , although this remains a rather controversial area . for instance vegf has been shown to stimulate hscs to produce collagen , to proliferate , and to migrate in  vitro . more recent studies , however , have shown that vegf is critical for reversal of fibrosis.56 , 57 the antifibrotic effects of vegf are most likely due to effects of vegf on sinusoidal endothelial cells rather than hscs . accordingly , the importance of vegf and angiogenesis to liver fibrosis is complicated and remains an evolving field . in addition to proangiogenic mediators , hypoxia increased the expression of receptors involved in hsc migration and activation . for instance , hypoxia increased the expression of the chemokine receptors ccr1 and ccr5 . in the presence of chemokines , activation of these receptors stimulates hsc migration . in support of a role for these receptors in the development of fibrosis two other receptors of interest were increased by hypoxia in hscs . these were interleukin-13 receptor 1 and adrenergic receptor 2b , which are activated by interleukin-13 and catecholamines , respectively . in  vitro , activation of these receptors on hscs stimulates collagen production , and in  vivo both these pathways have been shown to play an important role in liver fibrosis development.59 , 60 , 61 although up - regulation of some of the genes described here required hif-1 , up - regulation of some did not require hif-1 , suggesting an important role for hif-2 or potentially other hypoxia - regulated transcription factors in regulation of these genes . of particular interest , up - regulation of many of these genes by hypoxia only occurred in culture - activated hscs and did not occur in quiescent hscs , suggesting that hsc activation increases the sensitivity of hscs to hif - mediated gene expression changes . the mechanism by which this occurs is not known , but it may result from changes in histone modifications or dna methylation changes that modify access of hifs to the promoters of certain hif - regulated genes in activated hscs . in addition to the genes already described , hypoxia also increased expression of genes that have the potential to globally affect hsc function in a profound manner . jmjd6 regulates mrna spicing , and jmjd1a is a histone demethylase.62 , 63 up - regulation of jmjd1a by hifs could increase expression of a number of genes by modifying histone methylation , whereas up - regulation of jmjd6 could promote the formation of alternatively spliced mrnas that code for proteins with modified functions that promote fibrosis . in addition to jumoni proteins , hypoxia increased expression of regulator of g - protein signaling 2 ( rgs2 ) and rgs4 . these proteins act as gtpase - activating proteins for g protein  subunits , thereby decreasing signaling by gi , go , and gq subtypes . up - regulation of rgs proteins could substantially affect signaling by g protein - coupled receptors in hscs . the importance of this in regulating hsc function during the development of fibrosis remains to be determined . the various processes regulated by hifs in hscs that may contribute to fibrosis are summarized in figure  2 . although numerous genes were increased in hypoxic hscs , surprisingly few were decreased by hypoxia . two genes of particular interest that were decreased by hypoxia were hepatocyte growth factor ( hgf ) and 1-integrin . hgf is important for liver regeneration , and studies have shown that hscs are a source of hgf during regeneration.65 , 66 , 67 in addition to our study , corpechot et  al demonstrated in 2002 that hypoxia decreases hgf in hypoxic hscs . in this study , they proposed that decreased hgf expression in hypoxic hscs may contribute to liver regeneration failure in cirrhotic livers . alpha-1-integrin is a receptor for collagen , and studies have shown that collagen production by fibroblasts from 1-integrin knockout mice is enhanced , suggesting a role for this receptor in feedback inhibition of collagen production . the importance of the decrease in 1-integrin in hypoxic hscs , however , remains to be determined . furthermore , whether hif-1 contributes to the reduction in hgf and 1-integrin is not known . in consideration of the impact of hypoxia and hif activation on the function of hscs in  vitro , studies were conducted to evaluate the role of hif-1 in hscs in  vivo on the development of liver fibrosis . in these studies , hif-1 floxed mice were crossed with mice that express cre recombinase under the glial fibrillary acidic protein promoter , which , in the liver , is only expressed by hscs . treatment of control mice in this study with a single hepatotoxic dose of ccl4 increased the expression of types i , iii , and iv collagens by 48 hours after treatment . deletion of hif-1 in hscs completely prevented up - regulation of all three collagens in the liver at this time point . surprisingly , by 72 hours after ccl4 administration , the levels of type i collagen mrna and protein were much greater in the livers of mice in which hif-1 was knocked out in hscs . in addition , there were greater levels of -sma mrna and protein , indicating enhanced hsc activation . these results were quite contrary to in  vitro studies and suggested a potential role for hif-1 in limiting hsc activation and collagen production in  vivo after liver injury . further analysis , however , demonstrated that there was a failure of macrophages to become activated in the livers of these mice , which prevented removal of necrotic hepatocytes from the liver . we proposed that the persistence of necrotic cells most likely maintained hsc activation and enhanced production of collagen in mice deficient in hif-1 in hscs . interestingly , this phenotype is also observed in plasminogen and urokinase plasminogen activator knockout mice , where there is a failure to clear necrotic hepatocytes , leading to enhanced collagen production.70 , 71 , 72 accordingly , while hif-1 appeared to be important for collagen up - regulation early after injury , it also stimulated hscs to produce a hif-1dependent factor(s ) that activated macrophages , which is essential for phagocytic removal of necrotic hepatocytes . the mechanism by which this occurs is not known ; however , considering the similar phenotype in mice deficient in plasmin , it is possible that hif-1 activation in hscs modulates plasmin formation after injury . research over the past 16 years has provided important information on the role of hifs in the development of liver fibrosis . this work has provided evidence that hif-1 regulates a number of important profibrotic mediators in hepatocytes , hscs , and kupffer cells . it remains to be determined whether hif-1 activation in other cell types contributes to this disease . for instance , vegf , a key hif - target gene , is increased in cholangiocytes after bdl . whether this requires hif-1 is not known . hif-1 also regulates the function of various immune cell types , including t cells , natural killer t cells , and b cells , which may be important for fibrosis.74 , 75 , 76 , 77 furthermore , very little is known about the role of hif-2 in liver fibrosis and in which cells hif-2 is activated . finally , there is a clear link between hif-1 and hif-2 activation and the development of cancer . because cirrhosis can often progress to hepatocellular carcinoma , it would be interesting to determine whether hifs are an important driving force for the progression of cirrhosis to cancer . studies have shown that constitutive activation of hif-1 and hif-2 in hepatocytes produces hemangiomas in the liver , lending support for a role of hifs in cancer development in the liver . over the next several years , research should begin to fill these knowledge gaps . also , it is unclear whether pharmacologic inhibition of hifs would be an effective therapy for liver fibrosis . only one study has addressed this topic thus far . in a rat model of transarterial chemoembolization in which rats are treated with ccl4 and subjected to hepatic artery ligation , a putative hif-1 inhibitor called lw6 prevented progression of liver fibrosis when started 2 weeks after the initiation of liver disease . although these results are exciting , additional studies with other models of liver fibrosis and other well - characterized hif inhibitors need to be conducted before hifs are validated targets for liver fibrosis therapy .
liver fibrosis remains a significant clinical problem in the united states and throughout the world . 
 although important advances in the understanding of this disease have been made , no effective pharmacologic agents have been developed that directly prevent or reverse the fibrotic process . 
 many of the successes in liver fibrosis treatment have been targeted toward treating the cause of fibrosis , such as the development of new antivirals that eradicate hepatitis virus . for many patients , however , 
 this is not feasible , so a liver transplant remains the only viable option . 
 thus , there is a critical need to identify new therapeutic targets that will slow or reverse the progression of fibrosis in such patients . 
 research over the last 16 years has identified hypoxia - inducible factors ( hifs ) as key transcription factors that drive many aspects of liver fibrosis , making them potential targets of therapy . in this review , 
 we discuss the latest work on hifs and liver fibrosis , including the cell - specific functions of these transcription factors in the development of liver fibrosis .