Showing papers by "Raghu Kalluri published in 2010"

The Origin of Fibroblasts and Mechanism of Cardiac Fibrosis

Abstract: Fibroblasts are at the heart of cardiac function and are the principal determinants of cardiac fibrosis. Nevertheless, cardiac fibroblasts remain poorly characterized in molecular terms. Evidence is evolving that the cardiac fibroblast is a highly heterogenic cell population, and that such heterogeneity is caused by the distinct origins of fibroblasts in the heart. Cardiac fibroblasts can derive either from resident fibroblasts, from endothelial cells via an endothelial-mesenchynmal transition or from bone marrow-derived circulating progenitor cells, monocytes and fibrocytes. Here, we review the function and origin of fibroblasts in cardiac fibrosis.

Methylation determines fibroblast activation and fibrogenesis in the kidney

Abstract: Fibrogenesis is a pathological wound repair process that fails to cease, even when the initial insult has been removed. Fibroblasts are principal mediators of fibrosis, and fibroblasts from fibrotic tissues fail to return to their quiescent stage, including when cultured in vitro. In a search for underlying molecular mechanisms, we hypothesized that this perpetuation of fibrogenesis is caused by epigenetic modifications. We demonstrate here that hypermethylation of RASAL1, encoding an inhibitor of the Ras oncoprotein, is associated with the perpetuation of fibroblast activation and fibrogenesis in the kidney. RASAL1 hypermethylation is mediated by the methyltransferase Dnmt1 in renal fibrogenesis, and kidney fibrosis is ameliorated in Dnmt1(+/-) heterozygous mice. These studies demonstrate that epigenetic modifications may provide a molecular basis for perpetuated fibroblast activation and fibrogenesis in the kidney.

Origins of Cardiac Fibroblasts

Abstract: Cardiac fibroblasts play a critical role in maintenance of normal cardiac function. They are indispensable for damage control and tissue remodeling on myocardial injury and principal mediators of pathological cardiac remodeling and fibrosis. Despite their manyfold functions, cardiac fibroblasts remain poorly characterized in molecular terms. Evidence is evolving that cardiac fibroblasts are a heterogeneous population and likely derive from various distinct tissue niches in health and disease. Here, we review our emerging understanding of where cardiac fibroblasts come from, as well as how we can possibly use this knowledge to develop novel therapies for cardiac fibrosis.

Research Priorities in Hypertrophic Cardiomyopathy Report of a Working Group of the National Heart, Lung, and Blood Institute

Abstract: Hypertrophic cardiomyopathy (HCM) is a myocardial disorder characterized by left ventricular (LV) hypertrophy without dilatation and without apparent cause (ie, it occurs in the absence of severe hypertension, aortic stenosis, or other cardiac or systemic diseases that might cause LV hypertrophy). Numerous excellent reviews and consensus documents provide a wealth of additional background.1–8 HCM is the leading cause of sudden death in young people and leads to significant disability in survivors. It is caused by mutations in genes that encode components of the sarcomere. Cardiomyocyte and cardiac hypertrophy, myocyte disarray, interstitial and replacement fibrosis, and dysplastic intramyocardial arterioles characterize the pathology of HCM. Clinical manifestations include impaired diastolic function, heart failure, tachyarrhythmia (both atrial and ventricular), and sudden death. At present, there is a lack of understanding of how the mutations in genes encoding sarcomere proteins lead to the phenotypes described above. Current therapeutic approaches have focused on the prevention of sudden death, with implantable cardioverter defibrillator placement in high-risk patients. But medical therapies have largely focused on alleviating symptoms of the disease, not on altering its natural history. The present Working Group of the National Heart, Lung, and Blood Institute brought together clinical, translational, and basic scientists with the overarching goal of identifying novel strategies to prevent the phenotypic expression of disease. Herein, we identify research initiatives that we hope will lead to novel therapeutic approaches for patients with HCM. The epidemiology of HCM suggests that it is present in ≈1 in 500 adults.9 Because of the delay in phenotypic expression of the disease, HCM is not commonly recognized clinically in young children, but when it is, it is much more frequently recognized in males.10 This is likely due to greater penetrance in young males.11,12 HCM is underdiagnosed clinically in blacks and in women, yet …

Interstitial fluid: the overlooked component of the tumor microenvironment?

Abstract: Background: The interstitium, situated between the blood and lymph vessels and the cells, consists of a solid or matrix phase and a fluid phase, together constituting the tissue microenvironment. Here we focus on the interstitial fluid phase of tumors, i.e., the fluid bathing the tumor and stromal cells. Novel knowledge on this compartment may provide important insight into how tumors develop and how they respond to therapy. Results: We discuss available techniques for interstitial fluid isolation and implications of recent findings with respect to transcapillary fluid balance and uptake of macromolecular therapeutic agents. By the development of new methods it is emerging that local gradients exist in signaling substances from neoplastic tissue to plasma. Such gradients may provide new insight into the biology of tumors and mechanistic aspects linked to therapy. The emergence of sensitive proteomic technologies has made the interstitial fluid compartment in general and that of tumors in particular a highly valuable source for tissue-specific proteins that may serve as biomarker candidates. Potential biomarkers will appear locally at high concentrations in the tissue of interest and will eventually appear in the plasma, where they are diluted. Conclusions: Access to fluid that reliably reflects the local microenvironment enables us to identify substances that can be used in early detection and monitoring of disease.

Preeclampsia: 2-methoxyestradiol induces cytotrophoblast invasion and vascular development specifically under hypoxic conditions.

Abstract: Inadequate invasion of the uterus by cytotrophoblasts is speculated to result in pregnancy-induced disorders such as preeclampsia. However, the molecular mechanisms that govern appropriate invasion of cytotrophoblasts are unknown. Here, we demonstrate that under low-oxygen conditions (2.5% oxygen), 2-methoxyestradiol (2-ME), which is a metabolite of estradiol and is generated by catechol-o-methyltransferase (COMT), induces invasion of cytotrophoblasts into a naturally-derived, extracellular matrix. Neither low-oxygen conditions nor 2-ME alone induces the invasion of cytotrophoblasts in this system; however, low-oxygen conditions combined with 2-ME result in the appropriate invasion of cytotrophoblasts into the extracellular matrix. Cytotrophoblast invasion under these conditions is also associated with a decrease in the expression of hypoxia-inducible factor-1α (HIF-1α), transforming growth factor-β3 (TGF-β3), and tissue inhibitor of metalloproteinases-2 (TIMP-2). Pregnant COMT-deficient mice with hypoxic placentas and preeclampsia-like features demonstrate an up-regulation of HIF-1α, TGF-β3, and TIMP-2 when compared with wild-type mice; normal levels are restored on administration of 2-ME, which also results in the resolution of preeclampsia-like features in these mice. Indeed, placentas from patients with preeclampsia reveal lower levels of COMT and higher levels of HIF-1α, TGF-β3, and TIMP-2 when compared with those from normal pregnant women. We demonstrate that low-oxygen conditions of the placenta are a critical co-stimulator along with 2-ME for the proper invasion of cytotrophoblasts to facilitate appropriate vascular development and oxygenation during pregnancy.

Pre-eclampsia: connecting angiogenic and metabolic pathways.

Abstract: Pre-eclampsia is a hypertensive disease of pregnancy with a worldwide incidence of 5-8%. This review focuses on recent developments in pre-eclampsia research related to angiogenesis and metabolism. We first address the 'angiogenic imbalance' theory, which hypothesizes that pre-eclampsia results from an imbalance of factors that promote or antagonize angiogenesis, such as soluble fms-like tyrosine kinase (sFlt1), 2-methoxyestradiol (2-ME) and catechol-O-methyltransferase (COMT). Next, we analyze the association between pre-eclampsia and dysfunctional metabolism of both homocysteine and placental glycogen. We hope that illuminating some of the various connections existing between angiogenesis and metabolism in pre-eclampsia will facilitate the update or reconsideration of old models of pathogenesis.

Lack of Collagen XVIII/Endostatin Exacerbates Immune-Mediated Glomerulonephritis

Abstract: Collagen XVIII is a component of the highly specialized extracellular matrix associated with basement membranes of epithelia and endothelia. In the normal kidney, collagen XVIII is distributed throughout glomerular and tubular basement membranes, mesangial matrix, and Bowman's capsule. Proteolytic cleavage within its C-terminal domain releases the fragment endostatin, which has antiangiogenic properties. Because damage to the glomerular basement membrane (GBM) accompanies immune-mediated renal injury, we investigated the role of collagen XVIII/endostatin in this disorder. We induced anti-GBM glomerulonephritis in collagen XVIII α1-null and wild-type mice and compared the resulting matrix accumulation, inflammation, and capillary rarefaction. Anti-GBM disease upregulated collagen XVIII/endostatin expression within the GBM and Bowman's capsule of wild-type mice. Collagen XVIII/endostatin-deficient mice developed more severe glomerular and tubulointerstitial injury than wild-type mice. Collagen XVIII/endostatin deficiency altered matrix remodeling, enhanced the inflammatory response, and promoted capillary rarefaction and vascular endothelial cell damage, but did not affect endothelial proliferation. Supplementing collagen XVIII-deficient mice with exogenous endostatin did not affect the progression of anti-GBM disease. Taken together, these results suggest that collagen XVIII/endostatin preserves the integrity of the extracellular matrix and capillaries in the kidney, protecting against progressive glomerulonephritis.

Idiopathic pulmonary fibrosis is associated with endothelial to mesenchymal transition.

Abstract: Idiopathic pulmonary fibrosis (IPF) affects over 500,000 people in the United States and Europe alone. The disease is characterized by progressive obliteration of normal alveolar architecture and replacement by fibrotic tissue. The result is declining lung function, progressive dyspnea, and ultimately death within 3 to 5 years of diagnosis (1). Lung transplantation remains a therapeutic option for selected patients, as pharmacologic treatments have been unsuccessful in large, multi-centered studies. In the past, the inflammatory cascade has been regarded as vital to the development and progression of IPF. Therefore, pharmacologic therapies initially targeted inflammatory pathways with the goal of blunting the inflammatory response and thus impeding the development of fibrosis. However, only a small proportion of patients respond to anti-inflammatory therapies such as systemic corticosteroids, and the natural history of the disease remains largely unchanged. More recently, research in animal models has demonstrated that even in the absence of inflammation, fibrosis can be induced. Therefore, research has now included elucidating the mechanisms and mediators of fibrosis. The development of antifibrotic agents such as pirfenidone is encouraging; however, clinical data is lacking to justify its general use at this time (2, 3). At the heart of anti-fibrosis research has been determining the origin of fibroblasts as well as the signaling pathways that lead to an abnormal deposition of extracellular matrix by these fibroblasts. With regard to the former, historically, it was thought that fibroblasts in IPF arise solely from resident pulmonary mesenchymal cells. Interestingly, analysis of IPF fibroblasts reveals characteristics that are different from normal resident lung fibroblasts (4). In addition to resident pulmonary mesenchymal cells, fibroblasts may be derived from bone marrow progenitor cells that are recruited to injured tissue (5). Also, epithelial to mesenchymal transition (EMT) has been demonstrated as a potential source of fibroblasts in renal and pulmonary tissues (6–8). It is also recognized as playing a role in cancer metastasis. As the name implies, the process is characterized by a loss of epithelial markers (such as E-cadherin) and gain of mesenchymal markers such as a–smooth muscle actin (a-SMA) or fibroblast specific protein 1 (FSP-1). In addition, there is cytoskeletal rearrangement, cellular loss of apical-basal polarity, and loss of cell adhesion properties. The process is normal during embryonic development, but occurs as a pathological response to epithelial injury. Endothelial cells also display a significant amount of plasticity, especially in pathological conditions. Endothelial to mesenchymal transition (EndMT) is an example of such plasticity. Analogous to EMT, during EndMT, endothelial cells lose characteristic markers such as CD31 and gain mesenchymal markers (FSP-1 and a2SMA). EndMT is known to be a source of cancer-associated fibroblasts, accounting for as much as 40% in certain studies (9). Recently, endothelial cells were also demonstrated to play a role in cardiac fibrosis via EndMT (10). Furthermore, endothelial cells have been identified as sources of fibroblasts in models of diabetic nephropathy (11). Whether or not endothelial cells contribute to populations of fibroblasts during pulmonary fibrosis remains unclear. TGF-b is generally regarded as the key mediator for induction of EMT based upon in vitro and in vivo models. In pulmonary fibrosis, signaling pathways that interact with TGF-b to induce EMT include Wnt/b-catenin and Ras/ERK/MAPK (12). Signaling via TGF-b leads to phosphorylation of Smad2/Smad3 and their nuclear translocation. Transcriptional activation by Smad2/ Smad3 is important for the progression of EMT. Previous studies have demonstrated TGF-b and Notch to be important modulators of EndMT. In a murine model of cardiac fibrosis, signaling by TGF-b induced EndMT via Smad3 transcriptional activity (10). In the same model, inhibition of EndMT was possible by the addition of rhBMP-7. In pulmonary fibrosis, it remains unclear as to the signaling pathways that interact with TGF-b to induce EndMT. In this issue of the Journal, Hashimoto and colleagues (pp. 161–172) demonstrate that pulmonary capillary endothelial cells, through endothelial to mesenchymal transition, may serve as a source of fibroblasts in pulmonary fibrosis (13). Using the Tie2Cre/CAG-CAT-LacZ double transgenic mouse system, cells of endothelial origin constitutively express LacZ after Cre-mediated recombination, thereby labeling Tie2 promoter-activated cells. After bleomycin-induced lung injury, lung fibroblasts were isolated and tested for LacZ positivity (i.e., of endothelial origin). There were significantly more LacZ-positive fibroblasts in bleomycininjured lungs compared with saline-treated lungs. Of these LacZpositive fibroblasts in injured lungs, 15% were a-SMA–positive, indicating a myofibroblast phenotype. The remaining LacZpositive fibroblasts were a-SMA–negative, but positive for Col I, another marker of fibroblasts. Therefore, in this model of bleomycin-induced pulmonary fibrosis, endothelial cells can undergo mesenchymal transition to serve as sources of fibroblasts. In this model, cells from the bone marrow did not contribute significantly to the population of fibroblasts in the bleomycin-injured lungs. ‘‘Scattering’’ is a phenomenon in which the morphologic and gene expression changes that are characteristic of EMT are fully reversible upon removal of TGF-b. Although studies have documented the ability of lone TGF-b to induce irreversible EMT, others have noted that the administration of TGF-b alone, without activation of Ras/ERK/MAPK, induces scattering (7, 14). The current study suggests a dependence on Ras for completion of EndMT. Only treatment with TGF-b in combination with activated Ras induced a persistent morphological change and suppression of endothelial markers consistent with EndMT; treatment with TGF-b alone resulted in a scattering phenotype. In addition, only treatment with TGF-b in combination with activated Ras induced a-SMA expression; whereas neither TGF-b nor activated Ras alone were capable of doing so. These results provide evidence that Ras/MAPK, via TGF-b signaling, mediates completion of EndMT in a bleomycin model of pulmonary fibrosis (Figure 1). This is an interesting result in that the TGF-b and Ras interaction has mainly been demonstrated in cancer metastasis models of EMT but not in fibrosis models. The interpretation of this finding is difficult as this is an in The work in the authors’ laboratory is funded by DK55001 and funds from the Department of Medicine at the Beth Israel Deaconess Medical Center to R.K. and A.E.

Tumor Microenvironment Controls the Rate of Cancer Progression and Metastasis

Abstract: Tumors are unorganized organs that contain many different cell types that communicate with cancer cells (Kalluri NRC 2006). The central goal of our laboratory is to evaluate the role of these non-cancer cells in cancer progression and metastasis. Cancer progression significantly depends on the influence of many different host cells. Whether such host responses are recruited to control cancer progression or further aid in tumor growth (or both) is still unclear. This lecture will highlight the role of extra-cellular matrix, angiogenesis, and mesenchymal cells in cancer progression and metastasis, and discuss treatment strategies.

Mechanistic connection between inflammation and

Abstract: Regardless of the underlying etiology, most forms of chronic kidney diseases are characterized by progressive fibrosis as a final common pathway that eventually affects all substructures of the kidney with the final consequence of end-stage renal disease. Although there has been a great deal of research, comprehensive understanding of the pathogenetic mechanisms of kidney fibrosis remains uncertain and that hampers the development of effective therapeutic strategies. Fibrosis is a process of normal wound healing and repair that is activated in response to injury to maintain the original tissue architecture and functional integrity. However, prolonged chronic injurious stimuli may cause deregulation of normal processes and result in an excess deposition of extracellular matrix and fibrosis. It involves a complex multistage inflammatory process with inflammatory cell infiltration, mesangial and fibroblast activation, tubular epithelial to mesenchymal transition, endothelial to mesenchymal transition, cell apoptosis, and extracellular matrix expansion that is orchestrated by a network of cytokines/ chemokines, growth factors, adhesion molecules, and signal

Anti-glomerular Basement Disease: Goodpasture’s Syndrome

Abstract: Goodpasture’s syndrome is an exemplary rare lung and kidney disease that has led to significant discoveries in human biology. The initial observations that antibodies directed against glomerular basement membrane (GBM) caused glomerulonephritis stimulated evaluation of the components of the basement membrane, elucidation of the reticular collagen network, and identification of different types of collagen. The α3[IV] NC1 domain of type IV collagen is the antigenic epitope that initiates a complex autoimmune reaction culminating in the clinical manifestations of Goodpasture’s syndrome. Immunization with α3[IV] collagen has provided an experimental model that has led to fundamental discoveries into the genetic and immune processes precipitating and modulating autoimmune diseases. Initial evaluation of the autoimmune process focused on humoral mechanisms, but more recent studies suggest that the cellular immune process is activated and plays a key role in the pathogenesis of Goodpasture’s syndrome. Hemoptysis occurs in nearly all patients and renal histopathology demonstrates crescentic glomerulonephritis in the majority of cases. Immunofluorescence microscopy reveals the pathognomonic finding of linear deposition of IgG along with the glomerular capillaries and in the lung parenchyma. The diagnosis of anti-GBM disease is confirmed by the presence of circulating antibodies against basement membrane antigen in the correct clinical setting. Based on the pathogenetic mechanisms, therapeutic modalities include both induction and maintenance regimens: induction or initial therapy removes the pathogenic anti-GBM antibody by plasmapheresis and maintenance therapy reduces antibody production by immunosuppression. The prognosis of patients with anti-GBM disease depends on the level of renal dysfunction at presentation.