Biomedical subjects

David J Abraham

Publications and source records attributed to David J Abraham.

33 records · Page 2Linked to original sources

PubMed2004

Transgenic analysis of scleroderma: understanding key pathogenic events in vivo.

Modern molecular genetic methods have allowed better understanding of established mouse models of scleroderma and also facilitated the development of new and better defined mouse strains for investigating the pathogenesis of the disease. The best characterized scleroderma animal model is the type 1 tight skin mouse (Tsk1). Backcrossing these animals with other mutant strains has been informative. These experiments implicate the IL-4 ligand-receptor axis in the development of skin fibrosis. Parallel expression analysis of genes using microarrays has provided insight into novel mediators of fibrosis including the C-C chemokine MCP-3. Other experiments suggest that embryonically defined fibroblast-specific regulatory elements may be targets for activation in this model. The same lineage-specific elements have been used to selectively activate TGF beta signaling pathways in fibrosis to generate a novel model for scleroderma and also have been used to develop systems for ligand-dependent fibroblast-specific genetic recombination that will allow further analysis key candidate genes implicated in scleroderma pathogenesis. Better mouse models will improve understanding of this intractable rheumatic disease and can be expected to ultimately lead to improved treatments and outcome.

Animals↗

PubMed2004

Insights into the molecular mechanism of chronic fibrosis: the role of connective tissue growth factor in scleroderma.

Connective tissue growth factor (CCN2), a member of the CCN family of proteins, is a cysteine-rich matricellular protein. Connective tissue growth factor is not normally expressed in dermal fibroblasts unless induced. The most potent inducer of connective tissue growth factor thus far identified is transforming growth factor beta. Connective tissue growth factor, however, is constitutively overexpressed by fibroblasts present in skin fibrotic lesions, including scleroderma. The overexpression of connective tissue growth factor present in fibrotic lesions contributes to the phenotype of scleroderma in that connective tissue growth factor promotes matrix deposition, and fibroblast adhesion and proliferation. In animal models, whereas either transforming growth factor beta or connective tissue growth factor alone produce only a transient fibrotic response, connective tissue growth factor and transforming growth factor beta act together to promote sustained fibrosis. Thus the constitutive overexpression of connective tissue growth factor by fibroblasts present in fibrotic lesions would be expected to contribute directly to chronic, persistent fibrosis. This review discusses recent information regarding insights into connective tissue growth factor biology and, using scleroderma as a model system, the part connective tissue growth factor might play in fibrotic disease.

Animals↗

PubMed2004

TGF-beta signaling and the fibrotic response.

The cause of fibrotic diseases, pathologies characterized by excessive production, deposition, and contraction of extracellular matrix, is unknown. To understand the molecular basis of fibrotic disease, it is essential to appreciate how matrix deposition is normally controlled and how this process is dysregulated in fibrogenesis. This review discusses the current state of knowledge concerning interactions among the profibrotic proteins transforming growth factor-beta (TGF-beta), connective tissue growth factor (CTGF, CCN2), and ED-A fibronectin (ED-A FN) and the antifibrotic proteins tumor necrosis factor-alpha (TNF-alpha) and gamma-interferon (IFN-gamma).

Animals↗

PubMed2004

Regulation of collagen type I in vascular smooth muscle cells by competition between Nkx2.5 and deltaEF1/ZEB1.

A major component of the vessel wall of large arteries and veins is the extracellular matrix (ECM), which consists of collagens, elastin, and proteoglycans. Collagen type I is one of the most abundant of the ECM proteins. We have previously shown that the pro-collagen type I alpha 2 gene contains an enhancer which confers tissue-specific expression in the majority of collagen-producing cells, including blood vessels. In this paper, we delineate a specific vascular smooth muscle cell (vSMC) element: a 100-bp sequence around -16.6 kb upstream of the transcription start site that regulates collagen expression exclusively in vSMCs. Furthermore, we show that the expression is activated through the binding of the homeodomain protein Nkx2.5, which is further potentiated in the presence of GATA6. In contrast, this element was repressed by the binding of the zinc-finger protein deltaEF1/ZEB1. We propose a model of regulation where the activating transcription factor Nkx2.5 and the repressor deltaEF1/ZEB1 compete for an overlapping DNA binding site. This element is important in understanding the molecular mechanisms of vessel remodeling and is a potential target for intervention in vascular diseases where there is excessive deposition of collagen in the vessel wall.

Animals↗

PubMed2003

Constitutive connective tissue growth factor expression in scleroderma fibroblasts is dependent on Sp1.

Fibrotic diseases such as scleroderma (systemic sclerosis, SSc) are characterized by an excessive production of extracellular matrix and profibrotic proteins such as connective tissue growth factor (CTGF). In normal dermal fibroblasts, CTGF is not expressed unless induced by proteins such as tumor growth factor-beta (TGFbeta). Conversely, in fibroblasts cultured from fibrotic lesions CTGF mRNA and protein are constitutively expressed, even in the absence of exogenously added TGFbeta. Thus, studying the mechanism underlying CTGF overexpression in SSc fibroblasts is likely to yield valuable insights into the basis of the fibrotic phenotype of SSc and possibly other scarring disease. CTGF overexpression is mediated primarily by sequences in the CTGF promoter. In this report, we identify the minimal promoter element involved with the overexpression of CTGF in SSc fibroblasts. This element is distinct from the element necessary and sufficient for the induction of CTGF expression by TGFbeta in normal fibroblasts. Within this region is a functional Sp1 binding site. Blocking Sp1 activity reduces the elevated, constitutive levels of CTGF promoter activity and protein expression observed in SSc fibroblasts. Relative to those prepared from normal dermal fibroblasts, nuclear extracts prepared from SSc fibroblasts possess increased Sp1 binding activity. Removal of phosphate groups from nuclear extracts enhanced Sp1 binding activity, suggesting that phosphorylation of Sp1 normally reduces Sp1 binding to DNA. Thus, the constitutive overexpression of CTGF in SSc fibroblasts seems to be independent of TGFbeta signaling but dependent at least in part on Sp1.

Base Sequence↗

PubMed2003

Identification of PLOD2 as telopeptide lysyl hydroxylase, an important enzyme in fibrosis.

The hallmark of fibrotic processes is an excessive accumulation of collagen. The deposited collagen shows an increase in pyridinoline cross-links, which are derived from hydroxylated lysine residues within the telopeptides. This change in cross-linking is related to irreversible accumulation of collagen in fibrotic tissues. The increase in pyridinoline cross-links is likely to be the result of increased activity of the enzyme responsible for the hydroxylation of the telopeptides (telopeptide lysyl hydroxylase, or TLH). Although the existence of TLH has been postulated, the gene encoding TLH has not been identified. By analyzing the genetic defect of Bruck syndrome, which is characterized by a pyridinoline deficiency in bone collagen, we found two missense mutations in exon 17 of PLOD2, thereby identifying PLOD2 as a putative TLH gene. Subsequently, we investigated fibroblasts derived from fibrotic skin of systemic sclerosis (SSc) patients and found that PLOD2 mRNA is highly increased indeed. Furthermore, increased pyridinoline cross-link levels were found in the matrix deposited by SSc fibroblasts, demonstrating a clear link between mRNA levels of the putative TLH gene (PLOD2) and the hydroxylation of lysine residues within the telopeptides. These data underscore the significance of PLOD2 in fibrotic processes.

Amino Acid Sequence↗

PubMed2003

Fibroblast-specific expression of a kinase-deficient type II transforming growth factor beta (TGFbeta) receptor leads to paradoxical activation of TGFbeta signaling pathways with fibrosis in transgenic mice.

To better understand the role of disrupted transforming growth factor beta (TGFbeta) signaling in fibrosis, we have selectively expressed a kinase-deficient human type II TGFbeta receptor (TbetaRIIDeltak) in fibroblasts of transgenic mice, using a lineage-specific expression cassette subcloned from the pro-alpha2(I) collagen gene. Surprisingly, despite previous studies that characterized TbetaRIIDeltak as a dominant negative inhibitor of TGFbeta signaling, adult mice expressing this construct demonstrated TGFbeta overactivity and developed dermal and pulmonary fibrosis. Compared with wild type cells, transgenic fibroblasts proliferated more rapidly, produced more extracellular matrix, and showed increased expression of key markers of TGFbeta activation, including plasminogen activator inhibitor-1, connective tissue growth factor, Smad3, Smad4, and Smad7. Smad2/3 phosphorylation was increased in transgenic fibroblasts. Overall, the gene expression profile of explanted transgenic fibroblasts using cDNA microarrays was very similar to that of littermate wild type cells treated with recombinant TGFbeta1. Despite basal up-regulation of TGFbeta signaling pathways, transgenic fibroblasts were relatively refractory to further stimulation with TGFbeta1. Thus, responsiveness of endogenous genes to TGFbeta was reduced, and TGFbeta-regulated promoter-reporter constructs transiently transfected into transgenic fibroblasts showed little activation by recombinant TGFbeta1. Responsiveness was partially restored by overexpression of wild type type II TGFbeta receptors. Activation of MAPK pathways by recombinant TGFbeta1 appeared to be less perturbed than Smad-dependent signaling. Our results show that expression of TbetaRIIDeltak selectively in fibroblasts leads to paradoxical ligand-dependent activation of downstream signaling pathways and causes skin and lung fibrosis. As well as confirming the potential for nonsignaling receptors to regulate TGFbeta activity, these findings support a direct role for perturbed TGFbeta signaling in fibrosis and provide a novel genetically determined animal model of fibrotic disease.

Animals↗

PubMed2003

Connective tissue growth factor gene regulation. Requirements for its induction by transforming growth factor-beta 2 in fibroblasts.

In skin, the profibrotic protein connective tissue growth factor (CTGF) is not normally expressed. However, when skin cells are exposed to transforming growth factor-beta (TGF-beta), CTGF is induced in fibroblasts but not in epithelial cells. We have begun to investigate the requirements for the fibroblast-selective induction of CTGF by TGF-beta. Previously we found that this response was Smad-dependent. Now we show that protein kinase C and Ras/MEK/ERK are necessary for the TGF-beta induction of the CTGF promoter but not of a generic Smad-responsive promoter (SBE-lux). Induction of the CTGF promoter is antagonized by c-Jun or by MEKK1, suggesting that a proper balance between the Ras/MEK/ERK and JNK MAPK cascades is necessary for TGF-beta induction of CTGF. We identify the minimal CTGF promoter element necessary and sufficient to confer TGF-beta responsiveness to a heterologous promoter and show that a tandem repeat of a consensus transcription enhancer factor binding element, 5'-GAGGAATGG-3', is necessary for this induction. This element has not been previously shown to play a role in TGF-beta induction of gene expression in fibroblasts. Gel shift analysis shows that this sequence binds nuclear factors that are greatly enriched in fibroblasts relative to epithelial cells. Thus Smads, Ras/MEK/ERK, protein kinase C, and fibroblast-enriched factors that bind GAGGAATGG act together to drive the TGF-beta-mediated induction of CTGF in fibroblasts.

3T3 Cells↗

PubMed2003

Monocyte chemoattractant protein 3 as a mediator of fibrosis: Overexpression in systemic sclerosis and the type 1 tight-skin mouse.

OBJECTIVE: To determine the gene-expression profile in dermal fibroblasts from type 1 tight-skin (Tsk1) mice, and to examine the expression and potential fibrotic activity of monocyte chemoattractant protein 3 (MCP-3) in Tsk1 mouse and human systemic sclerosis (SSc) skin. METHODS: Complementary DNA microarrays (Atlas 1.2) were used to compare Tsk1 fibroblasts with non-Tsk1 littermate cells at 10 days, 6 weeks, and 12 weeks of age. Expression of MCP-3 protein was assessed by Western blotting of fibroblast culture supernatants, and localized in the mouse and human skin biopsy samples by immunohistochemistry. Activation of collagen reporter genes by MCP-3 was explored in transgenic mouse fibroblasts and by transient transfection assays. RESULTS: MCP-3 was highly overexpressed by neonatal Tsk1 fibroblasts and by fibroblasts cultured from the lesional skin of patients with early-stage diffuse cutaneous SSc. Immunolocalization confirmed increased expression of MCP-3 in the dermis of 4 of 5 Tsk1 skin samples and 14 of 28 lesional SSc skin samples, compared with that in matched healthy mice (n = 5) and human controls (n = 11). Proalpha2(I) collagen promoter-reporter gene constructs were activated by MCP-3 in transgenic mice and by transient transfection assays. This response was maximal between 16 and 24 hours of culture and mediated via sequences within the proximal promoter. The effects of MCP-3 could be diminished by a neutralizing antibody to transforming growth factor beta. CONCLUSION: We demonstrate, for the first time, overexpression of MCP-3 in early-stage SSc and in Tsk1 skin, and suggest a novel role for this protein as a fibrotic mediator activating extracellular matrix gene expression in addition to promoting leukocyte trafficking. This chemokine may be an important early member of the cytokine cascade driving the pathogenesis of SSc.

Adult↗

PubMed2003

The role of connective tissue growth factor, a multifunctional matricellular protein, in fibroblast biology.

Connective tissue growth factor (CTGF, CCN2), a member of the CCN family of proteins, is a cysteine-rich proadhesive matricellular protein that plays an essential role in the formation of blood vessels, bone, and connective tissue. As expression of this protein is potently induced by transforming growth factor-beta (TGFbeta), it has been hypothesized that CTGF mediates several of the downstream actions of TGFbeta. In particular, CTGF is profibrotic, as CTGF is overexpressed in fibrotic disease and synergizes with TGFbeta to promote sustained fibrosis in vivo. Over the last several years, key data regarding the developmental role and structure and function relationship of CTGF have emerged. In addition, increased information concerning the mechanisms underlying the control of CTGF expression in normal and fibrotic cells and the signal transduction pathways through which CTGF acts on cells has been uncovered. This review summarizes the current state of knowledge regarding CTGF biology.

Animals↗

PubMed2002

Prostacyclin derivatives prevent the fibrotic response to TGF-beta by inhibiting the Ras/MEK/ERK pathway.

The SMAD-mediated induction of connective tissue growth factor (CTGF), a fibroproliferative cytokine, by transforming growth factor (TGF)beta is required for the development of sustained fibrosis in humans. Here, we show that in fibroblasts, activation of the Ras/MEK/ERK pathway is required for the SMAD-mediated induction of CTGF by TGFbeta2. We then show that activation of protein kinase A (PKA) in fibroblasts is able to block Ras/MEK/ERK signaling and abolish the fibrotic response. Previously, we found that prostacyclin agonists were able to prevent the induction of CTGF in fibroblasts, and in patients with the fibrotic disease scleroderma. Here, we confirm the in vitro and in vivo antifibrotic effects of prostacyclin derivatives and show that these effects are due to PKA-dependent inhibition of the Ras/MEK/ERK pathway. Ras/MEK/ERK does not directly affect SMAD signaling. The coordinate and varied biological responses to TGFbeta are in part due to the interactions of signaling pathways within target cells. Specific inhibition of fibroblast Ras/MEK/ERK signaling might prevent fibrosis while leaving other physiological effects of TGFbeta unaltered.

Animals↗

PubMed2002

Myogenic cell proliferation and generation of a reversible tumorigenic phenotype are triggered by preirradiation of the recipient site.

Environmental influences have profound yet reversible effects on the behavior of resident cells. Earlier data have indicated that the amount of muscle formed from implanted myogenic cells is greatly augmented by prior irradiation (18 Gy) of the host mouse muscle. Here we confirm this phenomenon, showing that it varies between host mouse strains. However, it is unclear whether it is due to secretion of proliferative factors or reduction of antiproliferative agents. To investigate this further, we have exploited the observation that the immortal myogenic C2 C12 cell line forms tumors far more rapidly in irradiated than in nonirradiated host muscle. We show that the effect of preirradiation on tumor formation is persistent and dose dependent. However, C2 C12 cells are not irreversibly compelled to form undifferentiated tumor cells by the irradiated muscle environment and are still capable of forming large amounts of muscle when reimplanted into a nonirradiated muscle. In a clonal analysis of this effect, we discovered that C2 C12 cells have a bimodal propensity to form tumors; some clones form no tumors even after extensive periods in irradiated graft sites, whereas others rapidly form extensive tumors. This illustrates the subtle interplay between the phenotype of implanted cells and the factors in the muscle environment.

Animals↗

PubMed2002

Connective tissue growth factor: a new and important player in the pathogenesis of fibrosis.

Connective tissue fibrosis is the final common pathogenic process for almost all forms of chronic tissue injury. Whether caused by vascular dysfunction, inflammation, metabolic injury, trauma, or environmental agents, once initiated the fibrogenic process results in the progressive replacement of the normal tissue architecture with fibrotic lesions that eventually lead to organ compromise and failure. Fibrosis can be considered as a dysregulation in the normal tissue repair mechanism, resulting in severe tissue scarring. Fibrosis appears to be a consequence of linked processes, including the proliferation of resident fibroblast cell types, the increased production and deposition of extracellular matrix components, and the transition of fibroblasts into cells exhibiting a myofibroblast phenotype. Although transforming growth factor-beta (TGF beta) has long been regarded as a pivotal growth factor in the formation and maintenance of connective tissues and as a major driving influence in many progressive fibrotic diseases, attention has focused recently on the role of connective tissue growth factor (CTGF) in fibrosis. CTGF is selectively and rapidly induced in mesenchymally derived cells by the action of TGF beta. CTGF expression is increased in many fibrosing diseases. In addition, increasing evidence from in vivo and in vitro models of tissue remodeling and fibrosis suggest that CTGF may represent a downstream effector molecule of the profibrotic activities of TGF beta in the maintenance and repair of connective tissues and within fibrotic disease settings.

Animals↗

PubMed2002

CTGF expression in mesangial cells: involvement of SMADs, MAP kinase, and PKC.

BACKGROUND: The induction of excess matrix in renal fibrosis seems to be mediated, at least in part, by the transforming growth factor-beta (TGF-beta)-mediated induction of connective tissue growth factor (CTGF) in mesangial cells. METHODS: By examining CTGF protein and mRNA expression and promoter activity in the presence or absence of TGF-beta or inhibitors, the signaling pathways controlling basal and TGF-beta-induced CTGF expression in mesangial cells were investigated. RESULTS: TGF-beta enhances CTGF mRNA and protein expression in mesangial cells. Mutation of a consensus SMAD binding element in the CTGF promoter completely abolished TGF-beta-induced CTGF expression and reduced basal CTGF expression. The previously identified basal control element-1 (BCE-1) site, but not Sp1 contributes to basal CTGF promoter activity. Ras/MEK/ERK, protein kinase C (PKC) and tyrosine kinase activity also contribute to basal and TGF-beta-induced CTGF promoter activity in cultured mesangial cells. CONCLUSIONS: The TGF-beta-induction of CTGF in mesangial cells requires SMADs and PKC/ras/MEK/ERK pathways. SMADs are involved in basal CTGF expression, which presumably reflects the fact that mesangial cells express TGF-beta endogenously. TGF-beta also induces CTGF through ras/MEK/ERK. Inhibiting ras/MEK/ERK seems not to reduce phosphorylation (that is, activation) of SMADs, suggesting that SMADs, although necessary, are insufficient for the TGF-beta-stimulation of the CTGF promoter through ras/MEK/ERK. Thus, maximal TGF-beta induction of CTGF requires synergy between SMAD and ras/MEK/ERK signaling.

Animals↗

PubMedDate not supplied

Hypoxia-inducible factor 1 in lower limb ischemia.

In the Western world, peripheral vascular disease (PVD) has a high prevalence and is associated with high morbidity and mortality. More patients are presenting with critical limb ischemia (CLI), the end stage of PVD, because of an increased life expectancy owing to improved medical care. In a large percentage of these patients, lower limb amputation is still required, despite current advances in surgery and interventional radiology. Studies of ischemic skeletal muscles disclosed evidence of endogenous angiogenesis and adaptive skeletal muscle metabolic changes in response to hypoxia. Many of the genes responsible for these responses are regulated by hypoxia-inducible factor (HIF)-1. HIF-1, consisting of HIF-1alpha and HIF-1beta subunits, is a major transcription factor that functions as a master regulator of oxygen homeostasis that plays essential roles in cellular and systemic pathophysiology. HIF-1alpha expression and HIF-1 transcriptional activity increase exponentially as cellular oxygen concentration is decreased. More than 60 target genes that are transactivated by HIF-1 have been identified. Many of the target genes, such as vascular endothelial growth factor, have been studied extensively, especially in tumors. However, only recently that interest in HIF-1 is growing in relation to ischemic diseases. Most of the studies concentrated mainly on the angiogenic property of HIF-1. In contrast, there is a lack of information on the role of HIF-1 in skeletal muscle metabolic adaptive changes as the end-organ in PVD. This review aims to summarize our current understanding of HIF-1 roles and the therapeutic potential in PVD.

Angiogenic Proteins↗