1985 — 1990 |
Ramirez, Francesco B |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Chromosomal Organization of the Human Procollagen Genes @ Mount Sinai School of Medicine of Cuny
The primary goal of this proposal is to establish the chromosomal organization of the human procollagen genes. Up to date, at least five genetically distinct types of collagen have been identified: (a) Type I present mainly in skin, bones and tendons; (b) Type II major consituent of cartilage; (c) Type III found together with Type I in lung, liver, skin and vessels; and, (d) Types IV and V consituents of the basement membrane. All the different types are heterotrimers constituted of identical or similar Alpha-chains which are synthesized as precursor molecules with N- and C-propeptides (procollagens). The different Alpha-chains contain approximately 1000 residues and have a similar primary structure with the repeated amino acidic triplet -Gly, X, Y. Structural and functional features suggest that these nine (or more) genes may have evolved by multiple duplication from a common ancestral unit. These proteins are involved in human connective tissue inherited diseases such as: osteogenesis imperfecta, chondrodystrophies Marfan and Ehlers-Danlos syndrome. It is, therefore, of importance to establish the chromosomal organization of these genes, their linkage at the molecular level, and their association with other sequences in order to have a better understanding of their evolution, ontogenesis, tissue-specific distribution and expression in normal and pathological states. To achieve this goal we propose first to localize the procollagen genes using cloned cDNA specific for the proAlpha-chains of the different types. Second, to isolate from phage libraries collagen-like genes using cDNA and genomic probes under relaxed conditions of hybridization. Third, to establish the linkage among the different genes within a given cluster. Fourth, to characterize the genes with respect to their transcriptional boundaries, to flanking regulatory signals to their association with other genomic sequences which may play a role in their expression.
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0.946 |
1988 — 1992 |
Ramirez, Francesco B |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Control of Collagen Gene Expression in Lung Fibrosis @ Mount Sinai School of Medicine of Cuny
The primary objective of this proposal is to characterize at the molecular level the mechanisms and factors involved in the deranged expression of the human interstitial collagen genes in lung fibrosis. Toward this end we will: a) Establish the nature of cis-acting elements that modulate the tissue-specific expression of the collagens in mesenchymal cells, b) Analyze the changes in transcription that result from the treatment of cultured lung fibroblasts with a variety of growth factors and cell mediators, c) Monitor the temporal and cellular expression of the collagen genes during the pathogenic progression of the fibrotic process in bleomycinindued animals, and d) Explore the involvement of structural sequences in the modulation of collagen mRNA translation. To achieve these goals, the 5' regions of four human collagen genes will be subjected to detailed functional analyses using chimeric plasmids transfected into cultured normal, and induced lung fibroblasts. The activity of specific transcriptional factors will be assessed by a variety of molecular techniques on cloned DNA segments, and related to possible changes in chromatin structure. Regulatory molecules will be isolated and characterized with regard to their nature, and interaction with genomic elements. In situ hybridization will be employed to monitor the selective expression of collagen genes in tissues derived from a fibrotic animal model. Finally, the rate of translation of appropriate collagen constructs will be assayed in vitro, and in vivo following sequence manipulation. The long term thrust of this work is to elucidate the biological complexity and heterogeneity of lung fibrosis as it relates to collagen accumulation. In addition to collagen pathogenesis, these investigations will be of relevance to our general understanding of how cellular factors regulate the expression of eukaryotic genes.
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0.946 |
1990 — 1992 |
Ramirez, Francesco B |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Structure and Expression of Invertebrate Collagens @ Mount Sinai School of Medicine of Cuny
The extracellular framework of connective tissues in all metazoan phyla is provided for the most part by a group of structurally related proteins, the collagens. In addition to serving as supportive elements, these macromolecules greatly influence the spatial and ontogenic diversity of extracellular matrices, for they regulate a number of developmental programs and cellular activities, such as adhesion, proliferation and migration. Much information has been accumulated during the past several years on the structure, function and regulation of collagen genes in some vertebrate and invertebrate species. However, given the wide phylogenetic distribution of this gene family, a comprehensive evolutionary picture is still far from being attained. More importantly, the function and regulation of collagen during the crucial stages of early embryonic development has yet to be fully explored. It is the purpose of this application to address these important questions in an organism greatly amenable to the molecular analysis of early developmental processes, the sea urchin embryo. In support of these goals, we have recently isolated and partially characterized genes coding for two echinoid fibrillar procollagen chains. Specifically, we now plan to elucidate the entire structure of the proteins and corresponding genes in order to delineate further the evolution of the deuterostomial collagens. Patterns of collagen expression during embryonic development will be monitored using molecular and immunohistochemical techniques. Transgenic embryos and in vitro assays will be employed to identify sequences responsible for tissue- and stage-specific collagen gene expression, and to characterize their interaction with trans-acting factors. Specific trans-acting factors will be then purified and the cognate genes cloned and characterized. The long term thrust of this work rests on the premise that a better understanding of collagen biology and evolution will shed new light on animal morphogenesis and the relationships between structure and function of proteins.
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0.946 |
1991 — 1993 |
Ramirez, Francesco B |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Pathophysiology of Human Fibrillar Collagen Types @ Mount Sinai School of Medicine of Cuny
The mechanical properties of developing and mature organ systems are primarily determined by the deposition of fibrillar collagens in the extracellular matrix. It follows that structural defects in these macromolecules cause dominantly inherited conditions that adversely affect the integrity of bodily organs. In addition to their supportive function, collagens also play a dynamic role in a number of developmental programs and physiological processes, such as tissue remodeling and wound healing. Deregulated collagen biosynthesis is the hallmark of several pathological conditions, including fibrotic, inflammatory and arthritic diseases. It is the long-term goal of this application to elucidate the mechanisms underlying collagen pathophysiology. Toward this end, two major research themes are proposed. The first will explore the phenotypic consequences of collagen mutations in human conditions and in transgenic mice. The second will decipher the molecular circuitries that require collagen biosynthesis. Specifically, we will characterize type II collagen mutations in chondrodysplastic patients to confirm and extend the genetic and clinical understanding of this collagenopathy. We will utilize the transgenic mouse model to establish the function and pathogenesis of minor fibrillar collagen types. We will employ transfection experiments and DNA:protein binding assays to identify cis-acting regulatory elements that control the expression of collagen genes. Finally, we will clone the genes encoding the trans-acting factors that regulate collagen expression. The thrust of this work is to achieve a better understanding of connective tissue pathology as it relates to the function and biosynthesis of fibrillar collagen types.
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0.946 |
1993 — 1995 |
Ramirez, Francesco B |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Characterization of a Putative Microfibrillar Component @ Mount Sinai School of Medicine of Cuny
The 10-nm microfibrils are extracellular matrix aggregates of both elastic and nonelastic tissues. Very little is known about the composition and assembly of microfibrils, chiefly because of their highly insoluble nature. Among of the best characterized microfibrillar components is a 350-kDa glycoprotein, fibrillin. We and others recently cloned part of the fibrillin cDNA, mapped the gene to chromosome 15 (Fib 15), and established casual relationship between Fib 15 mutations and pleiotropic manifestations of Marfan syndrome. As a result of our cloning experiments, we also identified a fibrillin-like transcript whose gene resides on chromosome 5 (Fib 5). The second fibrillin locus was linked to congenital constractural arachnodactyly. This connective tissue disorder shares some of the skeletal manifestations of Marfan syndrome, in addition to displaying distinctive abnormalities of the joint and the external ear. We interpreted such a dual association between phenotypically related disorders and structurally related proteins as suggesting that the fibrillins may have distinct functions in different tissues, albeit both part of morphologically similar or identical aggregates. It is our long-term goal to test this hypothesis and, accordingly, in this application we describe experiments aimed at characterizing the structure, localization and pattern of expression of the Fib 5 gene product. Aside from elucidating new aspects of connective tissue pathophysiology, this descriptive work provides the basis of future investigations on the function and regulation of this newly discovered gene.
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0.946 |
1994 — 2005 |
Ramirez, Francesco B |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R37Activity Code Description: To provide long-term grant support to investigators whose research competence and productivity are distinctly superior and who are highly likely to continue to perform in an outstanding manner. Investigators may not apply for a MERIT award. Program staff and/or members of the cognizant National Advisory Council/Board will identify candidates for the MERIT award during the course of review of competing research grant applications prepared and submitted in accordance with regular PHS requirements. |
Pathophysiology of Fibrillar Collagen Types @ Mount Sinai School of Medicine of Nyu
DESCRIPTION: Correct spatial and temporal production of structurally normal collagens plays a critical role in the development and growth of the organism. Understanding the function of collagen proteins and the regulation of collagen genes is therefore an important prerequisite to decipher the etiopathogenesis of connective tissue disorders. The long-term goal of the applicant's research program is to characterize the genetic factors and molecular mechanisms responsible for tissue-specific assembly and function of collagenous macroaggregates. Toward this end, three major aims are proposed: 1) To elucidate how type I collagen production is modulated by antagonistic cytokines implicated in matrix remodeling. The components of a transcriptional complex of the human a2(I) collagen gene which mediates the action of transforming growth factor beta1 and tumor necrosis factor alpha will be cloned. 2) To characterize which DNA elements and nuclear factors control overlapapplicantng and mutually exclusive synthesis of types V and XI collagen chains. A combination of functional and DNA-binding assays will be used to dissect the cis-acting sequences and identify the trans-acting factors responsible for cell type-specific expression of the a2(V) and a1(XI) collagen genes. 3) To determine the function of the newly discovered type XIX collagen. The a1(XIX) collagen gene will be mutated by homologous recombination in mouse embryonic stem cells and the consequences for the development, growth and fitness of the animals will be examined. The results of these experiments will increase understanding of how collagens contribute to the assembly and function of the extracellular matrix in normal and diseased conditions.
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0.949 |
1995 — 1998 |
Ramirez, Francesco B |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Type Ii Procollagen and Chondrogenesis @ Mount Sinai School of Medicine of Cuny
Skeletogenesis is a complex and poorly understood biological process controlled by a variety of morphogenetic programs and cellular activities. Central to the harmonious formation and growth of the vertebrate skeleton is the proper development of cartilage. This is in turn the result of the correct spatiotemporal expression of several differentiation-specific genes, such as the one encoding the pro-alpha1 subunit of type II collagen. This extracellular glycoprotein is a critical contributor to the structural integrity of cartilaginous tissues, and to the growth and shape of skeletal bone. Type II collagen is also produced by epithelial and mesenchymal cells at pre- and non-chondrogenic sites of the developing vertebrate embryo. This early phase of gene expression is associated with the synthesis of a protein longer than the one made later in development and in the adult organism. The procollagen isoforms are the result of alternative splicing of exon 2 sequence. Exon 2 codes for the cysteine- rich motif of the N-propeptide, a sequence believed to exert a negative feedback control on collagen biosynthesis. The phylogenetic conservation of this tissue- and stage-specific pattern of gene expression strongly suggests a function other than structural for the long isoform of type II procollagen. Accordingly, it has been hypothesized that this macromolecule may be somehow implicated in establishing the subsequent pattern of skeletogenesis. It is the scope of this application to test the validity of this hypothesis using the power of the genetic approach. Toward this end, we propose to analyze the phenotype of transgenic mice expressing only one of the two pro-alpha1(II) collagen isoforms during the entire developmental process. In addition to providing new insights into the role of type II collagen in chondrogenesis, this work will also enhance our understanding of the pathogenesis of disorders that are directly or indirectly connected with the expression of this extracellular macromolecule.
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0.949 |
1995 — 1997 |
Ramirez, Francesco B |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Training Program in Cellular and Molecular Biology @ Mount Sinai School of Medicine of Nyu |
0.949 |
1996 — 1999 |
Ramirez, Francesco B |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Novel Microfibrillar Component @ Mount Sinai School of Medicine of Nyu
DESCRIPTION: The 10nm diameter microfibrils are extracellular matrix components that provide crucial physiomechanic properties to a wide variety of elastic and non-elastic tissues. Very little is known about the composition of the microfibrils, the way they are assembled, and the factors that regulate this process. The major and best characterized component of the microfibrils is fibrillin-1, a 350-kDa glycoprotein made of repeated EGF-like motifs interspersed among other cysteine-rich sequences. Mutations in fibrillin-1 are the cause of the clinical manifestations in individuals with Marfan syndrome. Work sponsored by this grant has led to the identification of another microfibrillar component structurally related to fibrillin-1, and thus termed fibrillin-2. It has also documented the diversified pattern of gene expression of the fibrillins during embryogenesis and within individual organ systems. Others have very recently corroborated the previous linkage data by identifying fibrillin-2 mutations in patients affected by Congenital Contractural Arachnodactyly, a condition that shares some of the skeletal manifestations of Marfan syndrome. Altogether, these observations support our original hypothesis or related but distinct roles for the fibrillins in the assembly and function of tissue-specific matrices. In this competing continuation, we expand further the formulation of this early hypothesis. Accordingly, we propose that transcriptional programs which modulate the developmental production of fibrillin proteins are ultimately responsible for the tissue-specific function of microfibrillar aggregates and elastic networks. Experiments described in this proposal are aimed at elucidating the function of fibrillin-2 in matrix assembly and maintenance, and at characterizing the spatio-temporal regulation of the fibrillin genes. Like int he past, they will closely interact with colleagues working on the ultrastructural aspects of microfibril assembly and elastogenesis, and the molecular genetics of human connective tissue disorders. Collectively, results of these studies will enhance our understanding of microfibril biology; shed new light on macromolecular assemblies in normal and diseased states; and clarify the range of contributions of the extracellular matrix to animal morphogenesis and development.
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0.949 |
1998 — 2001 |
Ramirez, Francesco B |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Collagen Xi Isoforms in Cartilage and Bone Physiology @ Mount Sinai School of Medicine of Nyu
DESCRIPTION (Adapted from the Applicant's Abstract): The hierarchical assembly of proteoglycan and collagen types II, IX and XI networks leads to the formation of an organized cartilage matrix. Mutations of the components of the collagen fibrils are associated with a wide spectrum of clinical conditions that range from mild osteoarthritis to lethal chondrodysplasia. Genetic evidence in the mouse has revealed that incorporation of collagen XI in the heterotypic fibrils is a prerequisite for the development and differentiation of the skeleton. It has also suggested that this role is mediated by the alpha subunit through a mechanism yet to be determined. It is the main goal of this application to clarify the nature of this mechanism. Previous work has documented that the structural diversity of the N-terminal domain of alpha1(XI collagen is due to tissue- and stage-specific alternative splicing. This evolutionarily conserved pattern gives rise to four major isoforms, each of which is predominantly expressed at different times of differentiation and in distinct areas of cartilage. This proposal wishes to test the hypothesis that the alpha1(XI) isoforms modulate fibril growth, stabilize the matrix, and delineate areas destined to become bone. Toward this end, the structural-functional relationships of the polymorphic sequences will be examined within the context of the developing mouse. The long-term goal of the project is to elucidate the contribution of collagen XI to matrix function and to the physiomechanical properties of cartilage and bone. It is suggested (by the applicant) that this information will advance our understanding of the pathogenesis of disorders that impair skeletal function.
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0.949 |
2001 — 2002 |
Ramirez, Francesco B |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Mechanisms of Ethanol Induced Liver Fibrogenes @ Mount Sinai School of Medicine of Nyu
Alcohol-induced liver cirrhosis is one of the major causes of death worldwide. Strong evidence has established that acetaldehyde, ethanol's first metabolite, is fibrogenic per e and enhances type I collagen production by hepatic stellate cells. Despite major efforts by several laboratories, relatively little is known pertaining the molecular events underlying this stimulatory effect. Thus, it is our long-term goal to integrate cellular and molecular events into a unified picture that explains how acetaldehyde stimulates type I collagen gene transcription in the liver. The specific aims of this proposal: 1) To characterize the cis-regulatory elements and trans-acting factors, active in hepatic stellate cells of human origin, mediating acetaldehyde-induced human alpha2(I) collagen gene up-regulation; 2) To elucidate key molecular mechanisms by which acetaldehyde modulates the activity and/or binding of the transcription factors that interact with the acetaldehyde- responsive element of the human alpha2(I) collagen gene; and 3) To determine whether acetaldehyde stimulates human alpha2(I) collagen gene expression directly, or through an autocrine loop involving enhanced production and/or activation of transforming growth factor-beta in human hepatic stellate cells. A better characterization of the molecular events underlying enhanced COL1A2 gene expression in hepatic stellate cells, will enhance our understanding of the pathophysiology of ethanol- induced liver fibrosis, and will allow in the near future, to develop new therapies aimed at counteracting the devastating effects of this disease.
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0.949 |
2007 — 2011 |
Ramirez, Francesco B |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Pathophysiology of Basement Membrane Zone Collagens @ Mount Sinai School of Medicine of Nyu
[unreadable] DESCRIPTION (provided by applicant): We seek to elucidate how collagen molecules participate in the assembly of higher order tissue structure and implicitly, how collagen dysfunctions contribute to the genesis and progression of connective tissue disorders. Collagens are the most abundant and diverse components of the connective tissue and thus, the major structural determinants of organ formation and function. Stage and tissue-specific interactions among collagens, other matrix components, surrounding cells and signaling molecules are responsible for the variety of forms and functions of the developing and mature connective tissue. In spite of much progress, there is still a significant gap in our understanding of the mechanisms that translate the structural and cellular interactions of collagen molecules into the physiological properties of connective tissues. The lack of this basic science information in turn hampers our ability to model effective new therapies for a variety of human diseases in which organ function is severely or irreversibly impaired. Based on past discoveries and exciting new data, we hypothesize that timely deposition of collagen XIX in selected basement membrane zones (BMZ) specifies their organization and instructs tissue differentiation. BMZ are morphologically defined entities that consist of BM scaffolds interposed between the cells and interstitial matrices. BMZ play both structural and instructive role in tissue formation, maintenance and remodeling. Mutations in BMZ-associated collagens result in clinical manifestations as diverse as skin blistering, cardiovascular dysfunctions, ocular degeneration, kidney failure, hemorrhagic stroke, and muscle degeneration. In contrast to the cell-BM interface, information about the molecular interconnections between the BM and the underlying stroma is still primitive. Our genetic studies in mice have recently implicated collagen XIX as a potential new component of the molecular network that organizes the architecture of specialized BMZ. They have also suggested that assembly of a collagen XlX-rich matrix promotes cell differentiation. It is the main objective of the present application to fully characterize the role of collagen XIX in organ physiology, as the means to advance knowledge of the mechanisms that specify tissue architectures and functions. We therefore propose to characterize the structural and instructive roles of collagen XIX by studying the structural properties and molecular interactions that contribute to BMZ assembly and function, and tissue differentiation and growth. Clinical relevance: The proposed studies will generate new basic science information that will improve our understanding of the genesis and progression of connective tissue disorders, with potential benefits to the clinical management and therapy of many of these debilitating and often lethal conditions. [unreadable] [unreadable] [unreadable]
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0.958 |
2008 — 2009 |
Ramirez, Francesco B |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Experimental Models of Scleroderma Pathogenesis @ Mount Sinai School of Medicine of Nyu
[unreadable] DESCRIPTION (provided by applicant): We seek to advance knowledge of the mechanisms responsible for excessive collagen production by scleroderma fibroblasts. Scleroderma (Systemic Sclerosis; SSc) is a clinically heterogeneous and poorly understood disorder of the connective tissue that is characterized by vascular, immune/inflammatory and fibrotic manifestations. In spite of much effort, there are currently neither effective therapies nor reliable diagnostic/prognostic tools to manage the progression of tissue fibrosis, the main cause of morbidity and mortality in SSc. We base this application on exciting new evidence that circulating auto-antibodies against the PDGF receptor (PDGFR) are causally associated with the pro-fibrotic phenotype of SSc fibroblasts. Specifically, we have shown that anti-PDGFR auto-antibodies are only present in SSc sera, and that they can convert healthy fibroblasts into SSc-like cells. Identified markers of fibroblasts activation include excessive reactive oxygen species (ROS) production, Ha-Ras stabilization, increased PDGFR levels, and amplified collagen I transcription through TGF-2 independent activation of Smad3. We hypothesize a disease mechanism whereby anti-PDGFR auto-antibodies trigger a ROS-driven signaling cascade that promotes unopposed production of TGF-2, PDGFR, and collagen. Our model is in line with previous findings of elevated PDGFR in association with deregulated TGF-2 activity in SSc dermal lesions and explanted fibroblasts. It is also consistent with evidence of functional relationships between Ras and Smad signals, and reciprocally positive regulation of PDGF and TGF-2 signals. We propose to characterize key downstream events and pathogenic properties of the anti-PDGFR auto-antibodies. Whereas the former aim will be pursued using traditional cell culture systems, the latter one will employ a novel animal model of skin fibrosis. Completing these studies will improve our understanding of SSc pathogenesis, and may also identify novel therapeutic opportunities to manage tissue fibrosis more effectively. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: Scleroderma is a relatively rare and often fatal disorder that affects mostly adult women. Death occurs from end-stage organ failure caused by massive fibrosis in the connective tissue of skin, vessels and internal organs. There are currently neither effective therapies nor reliable prognostic tools to manage disease progression, mostly due to our limited understanding of scleroderma pathogenesis. We have recently discovered that circulating auto-antibodies against PDGFR receptor (PDGFR) may represent a main determinant of the pro-fibrotic phenotype of scleroderma fibroblasts. The present application seeks to further characterize key mechanisms and pathogenic properties of anti-PDGFR auto-antibodies, with the long-term goal of developing new targeted therapies against this devastating disorder. [unreadable] [unreadable] [unreadable]
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0.958 |
2009 — 2012 |
Ramirez, Francesco B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Novel Therapeutic Targets and/or Prognostic Markers in Marfan Syndrome @ Icahn School of Medicine At Mount Sinai
Marfan syndrome (MFS) is a congenital disorder of the connective tissue that is caused by mutations in fibrillin-1, the major structural component of extracellular microfibrils. We have originally hypothesized and subsequently demonstrated that flbrillin-1 mutations impair the sequestration of latent TGFp complexes in the extracellular matrix with deleterious consequence to cell performance. This seminal discovery has led to the realization that TGFp blockade is a productive new strategy to mitigate systemic manifestations in mouse models of MFS and perhaps in human pafients. We now present data that implicate MAPKs and BMPs as addifional contributors to organ-specific abnormalifies in mouse models of MFS. These exciting new findings are consistent with the emerging role of microfibrils in the extracellular storage of several TGFp superfamily members. They are also in line with the notion that TGFp and BMPs can signal through MAPK-mediated pathways and that MAPKs can influence Smad activity in response to environmental stress and tissue injury. We therefore hypothesize that flbrillin-1 mutations trigger a series of matrix-dependent events that disrupt the physiological balance of TGFp and BMP signaling in individual tissues, and that improper stimulafion of p38 MAPK activity (through TGFp and/or stress-response signals) exacerbates this disease-causing process Accordingly, the main focus of the present project is to evaluate the potential role of p38 and BMPs in the pathogenesis of aortic aneurysm and skeletal deformities in mouse models of progressively severe MFS Specifically, we propose to: Aim 1: Interrogate the contribufion of p38 activity to aortic aneurysm in Fbn1 mutant mice that are also defective in non-canonical TGFp signaling or are systemically treated with inhibitors of p38 or Smad2/3 signaling. Aim 2: Evaluate the role of unbalanced TGFp and BMP signaling in bone overgrowth using mice in which Fbnl deficiency is paired with loss of TGFp or BMP type II receptors, or with over-expression of a BMP antagonist. The proposed studies will complement and be informed by parallel investigations on the contribution to aortic aneurysm of ERK1/2 signaling (Project 1), latent TGFp activators (Project 2) and a structurally abnormal extracellular matrix (Project 3). Additionally, the unique reagents and imaging services of Core B will provide critical support to the work. Collectively, our efforts will shed new light on the complexity of the physiological roles that fibrillin-rich microfibrils play in organ formafion and function, in addition to providing evidence-based opportunities for therapeutic intervention in MFS and related disorders of the connective tissue.
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0.958 |
2013 |
Ramirez, Francesco B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Developmental Basis of Aneurysm in Marfan Syndrome and Therapeutic Implication @ Icahn School of Medicine At Mount Sinai
Marfan syndrome (MFS) is a common disorder caused by mutations in the gene encoding the matrix protein fibrillin-1. Our prior work has shown that many manifestations of MFS, including aortic aneurysm, valve disease, emphysema and skeletal muscle myopathy, are caused by excessive activation of and signaling by the TGF beta family of growth factors and can be attenuated by TGFbeta blockade in mouse models. The prevailing view has been that MFS manifests abnormal behaviors of normal cells due to alterations in their extracellular environment. We now present evidence for abnormal cells within the aortic wall of adult MFS mice that have undergone a TGFbeta-dependent permanent transition in identity and character during early development due to a process termed endothelial-to-mesenchymal transition (EnMT). After transition, resulting myofibroblasts exhibit many deleterious behaviors including high TGFbeta signaling, angiotensin II (Angll)-dependent fibrosis, and high expression of matrix-degrading enzymes. The major hypotheses to be tested in this work are that EnMT-derived cells drive progression of disease and that EnMT continues to populate the ascending aorta during postnatal life in disease states. Using mouse models, we will determine the pathways that drive EnMT in the aorta of fibrillin-1 deficient mice and will purify EnMT-derived cells, allowing identification of their deleterious behaviors and exploration of strategies to tame them. Currently, we can envision at least 9 different therapeutic agents that will theoretically prevent ongoing EnMT and/or modulate the nonproductive performance of myofibroblasts resident within the aortic wall at the time of initiation of treatment. These will be tested in genetically defined and validated mouse models of MFS. Remarkably, a number of these agents are already in clinical use for other indications, suggesting the potential for rapid translation to people with MFS. Our current data suggest a developmentally-imposed fixed alteration in cellular identify in the prediposition for apparently acquired late-onset phenotypes in MFS, This paradigm represents a novel way of thinking about genetic predisposition, aids in the elucidation of therapeutic limitations and opportunities, and will likely prove relevant to other conditions.
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0.958 |
2013 — 2014 |
Ramirez, Francesco B |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Structural Microenvironment of Bone Marrow Stem Cells @ Icahn School of Medicine At Mount Sinai
DESCRIPTION (provided by applicant): The definition of adult stem cell niches in the bone marrow (BM) and their regulation by extrinsic factors is a top research priority in tissue engineering and skeletal regenerative medicine, as well as a critical aspect of hematopoietic function and its reconstitution after marrow ablation therapy. Unlike the wealth of information regarding the cell types, cell-cell interactions and soluble signals that specify functionally discrete BM microenvironments, significantly less is known about the role of the extracellular matrix (ECM) in niche function to the detriment of developing more effective stem cell-based therapies. Our preliminary findings are the first to indicate involvement of fibrillin-1, a unique ECM protein that regulates the spatial organization and physical properties of tissues as well as the bioavailability of endogenous (local) TGF¿ family signals. This discovery raises the exciting possibility that a structural component of the marrow matrix controls the production of bone, blood and immune cells by determining the physical microenvironment of resident stem cells and by coordinating regulatory signals within it. Fibrillin-1 is the mutated protein in Marfan syndrome (MFS), whose pleiotropic manifestations include progressive bone loss (osteopenia). We previously demonstrated that osteopenia in MFS mice reflects perturbed bone remodeling due to impaired calibration of local anabolic and catabolic signals. Ongoing investigations have implied that fibrillin-1 is an essential component of the BM microenvironment that specifies the performance of mesenchymal stem cells (MSC) and hematopoietic stem cells (HSC). The goal of this high risk/high reward R21 application is to answer the following two questions: (a) What are the phenotypic consequences of fibrillin-1 deficiency for BM-based osteogenesis and hematopoiesis? ; (b) Does fibrillin-1 coordinate MSC-supported HSC performance? Accordingly, we propose to: (Aim 1) Characterize impaired MSC activity and bone loss progression in mice lacking fibrillin-1 in the appendicular skeleton and to provide a full account of hematopoietic abnormalities in these mutant animals; and (Aim 2) Validate the role of fibrillin-1 in coordinating MSC-supported HSC differentiation using mice with conditional Fbn1 inactivation in a specific stromal cells population. By demonstrating that fibrillin-1 is an indispensable functional component of BM niches, the experiments will establish the basis for future interrogation of the mechanism mediating fibrillin-1 regulation of marrow niches; and by implicating fibrillin-1 in immune system function, they may also lead to a radically new understanding of the cellular events responsible for aortic disease progression in MFS with unanticipated opportunities for therapy. Overall, this highly innovative proposal is expected to yield novel insights into age-related bone loss, advance fundamental knowledge of stem cell biology, impact a variety of translational applications in regenerative medicine, and perhaps improve the clinical management of life- threatening aortic manifestations in MFS.
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0.958 |
2015 — 2018 |
Ramirez, Francesco B |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Characterization of a Novel Pathway of Aortic Aneurysm Formation. @ Icahn School of Medicine At Mount Sinai
? DESCRIPTION (provided by applicant): Aortic aneurysms are common pathologies often associated with tear (dissection) and rupture of the vessel wall. Despite substantial research effort, therapeutic options to prevent death from ruptured aortic aneurysm remain limited. In contrast to abdominal aortic aneurysm (AAA), thoracic aortic aneurysm (TAA) is frequently caused by mutations in proteins that promote arterial tissue integrity and homeostasis. Characterization of pathogenic mechanism of aortic aneurysms in genetically altered or experimentally manipulated mice has identified both common and unique disease determinants, in addition to raising some controversies. Unresolved issues relevant to the development of new therapeutic strategies include the mechanisms of improper signaling by angiotensin II type I receptor (AT1r) and by TGF? in the dilating aorta. We propose to use a monogenic mouse model of TAA to address these key issues with the long-term goal of identifying new biological targets for drug therapy. The premise of our studies rests on strong preliminary evidence indicating that wall stress is a primary activator of persistent AT1r signaling and that the homeodomaininteracting protein kinase 2 (Hipk2) is an unsuspected stimulator of TGF? hyperactivity activated by AT1rinduced reactive oxygen species (ROS) signaling. We therefore hypothesize that progressive hemodynamic load on a biomechanically impaired aorta triggers stress signals that are sensed and mediated by AT1r via ROS to augment TGF? activity in part through Hipk2 action. We will test our hypothesis by monitoring TAA modifications in MFS mice with different genetic and pharmacological manipulations of the AT1 and AT2 receptors (Aim 1a and Aim 1b); by analyzing the molecular responses of healthy and mutant aortic strips to ex vivo stretching (Aim 1c); by elucidating the pathogenic contribution of Hipk2 to TAA formation in relationship to improper AT1r and TGF? signaling (Aim 2a); by supporting the in vivo findings with molecular and biochemical analyses of Hipk2 dysregulation in mutant vascular smooth muscle cell cultures (Aim 2b); and by validating mouse derived evidence of Hipk2related molecular abnormalities in aortic tissue specimens isolated from MFS patients (Aim 2c). The proposed work is expected to shed new light on the structural, molecular and mechanical determinants of aortic aneurysms and identify potential drug targets to blunt arterial disease. As such, our work is predicted to advance basic scientific knowledge, improve clinical care and guide translational applications for this life-threatening group of arterial disease conditions.
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0.958 |
2016 — 2020 |
Ramirez, Francesco B |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Tendon-Dependent Control of Longitudinal Bone Growth @ Icahn School of Medicine At Mount Sinai
? DESCRIPTION (provided by applicant): A disproportionate increase of longitudinal bone growth that causes serious malformations of the limbs, anterior chest and spine is the clinical hallmark of patients afflicted with Marfan syndrome (MFS), a connective tissue disease caused by mutations in the extracellular matrix (ECM) protein and TGF? regulator fibrillin-1. Our preliminary studies of mice with tissue-specific ablated Fbn1 gene activity have revealed an unsuspected causal relationship between tendon/ligament (T/L) dysfunction and longitudinal bone overgrowth (LBO). Specifically, we found that (1) Fbn1 inactivation in T/L cells was necessary and sufficient to promote linear bone overgrowth associated with dysregulated growth plate (GP) gene expression; (2) fibrillin-1-deficient tendons displayed abnormal tissue architecture and impaired mechanical properties, particularly at bone- insertion sites; (3) the relative amount of fibrillin-1 correlated with discrete changes in tendon mechanics; (4) tendon-derived stem/progenitor cell (TSPC) cultures deficient for fibrillin-1 differentiated improperly as result of increased latent TGF? activation; and (5) ectopic tendon calcification of fibrillin-1-deficient tendons was commonly observed. We therefore hypothesize that fibrillin-1 assemblies normally restrict GP-driven linear growth of neighboring bones by specifying the mechanical properties of tendons through the control of ECM organization and TGF?-regulated TSPC differentiation. Accordingly, the scope of our proposal is two-fold; first, to characterize how fibrillin-1 deficiency translates into tendon dysfunction and tendon-associated LBO, and second, to establish how local TGF? hyperactivity in tendons promote tissue degeneration thereby leading to excessive linear growth of the adjacent, structurally normal bones. To this end, we will characterize the expression of molecular and cellular determinants of tendon development and maturation in mice deficient for fibrillin-1 in T/L matrices, in addition to employing computational approaches to identify probable disease-causing molecular abnormalities in the GP of these tendon-defective animals (Aim 1); apply data-driven statistical models to determine how graded fibrillin-1 deficiencies correlate with tendon mechanics and associated LBO (Aim 2); and assess whether systemic TGF? neutralization modifies tendon pathology and LBO severity in fibrillin-1-deficient mice (Aim 3). The results of these investigations are expected to substantially advance our limited understanding of tendon function in health and disease and implicitly, of the cellular, molecular and tissue factors that coordinate the postnatal growth of musculoskeletal tissues.
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0.958 |
2017 — 2020 |
Iyengar, Srinivas Ravi V Ramirez, Francesco B |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mouse Models For Systems Therapeutics Degenerative Diseases @ Icahn School of Medicine At Mount Sinai
Project summary Thoracic aortic aneurysms (TAA) are life-threatening pathologies characterized by progressive dilation associated with smooth muscle cell dysfunction and destructive extracellular matrix remodeling that ultimately lead to tear and rupture of the vessel wall. Current management of TAA relies on early detection by routine imaging and prophylactic repair by surgical procedures. The main goal of this proposal is to use systems therapeutics as an unbiased tissue-level strategy to identify combinatorial treatments with repurposed drugs that can efficaciously target TAA-related signaling pathways in mice with early onset, progressively severe Marfan syndrome (MFS). This genetic model of TAA was chosen because the mutated protein (fibrillin-1) regulates several key aspects of arterial function and homeostasis, including tissue integrity, endothelial cell mechanotransduction, and angiotensin II and TGF? signaling. Our proposal is organized into two specific aims that combine experimental and computational approaches to elucidate the pathogenic contributions of multiple signaling pathways and cell types in the fibrillin-1-deficient aortas of MFS mice (Aim 1); and predict and validate combinatorial drug treatments targeting disease-associated signals within and across distinct wall compartments (Aim 2). Our system therapeutics strategy is expected to transform MFS from a deadly arterial disease that requires surgical intervention to a chronic condition that can be managed medically.
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0.958 |
2018 — 2021 |
Ramirez, Francesco B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Characterization of Altered Mechanosensing in Mouse Models of Ecm-Induced Taa @ New York University School of Medicine
Project summary Thoracic aortic aneurysms (TAAs) are a group of life-threatening conditions driven by complex pathophysiological interactions among different cell types, distinct extracellular matrix (ECM) compartments and multiple biochemical signals that, together, predispose the vessel wall to dissection and rupture. We hypothesize that, irrespective of the underlying cause, dysfunctional mechanobiology is a shared mechanism of TAA development and consequently, that molecules sensing, transducing or countering mechanical stress may represent potential new targets for drug therapy. This PPG focuses on identifying key disease-related cellular responses that are triggered either by experimentally increased blood pressure on a structurally normal tissue or by physiological hemodynamic load on a genetically deficient matrix. Project 1 employs a mouse model of progressively severe Marfan syndrome (MFS) to characterize the latter mechanism of arterial disease. This genetic model of TAA was chosen because the mutated protein (fibrillin-1) regulates several key aspects of arterial function and homeostasis, including tissue integrity, endothelial cell mechanotransduction, angiotensin II (AngII) type I receptor (AT1r) activity and TGF? signaling. Our proposal is organized into two specific aims that combine genetic, biomechanical and computational approaches to elucidate the primary triggers and downstream mediators and targets of AngII-dependent and AngII-independent AT1r signaling in distinct aortic compartments of MFS mice (Aim 1), and to evaluate how incremental loss of ECM integrity influences the biological responses of aortic cells to induced hypertension (Aim 2). Expected findings will inform, extend or complement the studies pursued by other PPG projects, which collectively will provide a new tissue-level understanding of the molecular factors and cellular events responsible for TAA onset and progression in a validated mouse model of lethal MFS.
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0.905 |