Anie Philip

Angiogenesis and growth factor expression in a model of transmyocardial revascularization.

BACKGROUND The mechanism by which transmyocardial revascularization (TMR) exerts a beneficial effect remains unknown. We hypothesize that the myocardial punctures of TMR cause a myocardial injury, leading to an angiogenic response mediated by a number of growth factors. METHODS Fifty-three rats underwent ligation of the left coronary artery. Group I (n = 25) served as controls, whereas group II (n = 28) underwent concomitant TMR by the creation of six transmural channels with a 25-gauge needle in the ischemic zone. Surviving animals in both groups were sacrificed at intervals of 1, 2, 4, and 8 weeks (n = 5 in each subgroup). Immunohistochemistry in the infarct areas was performed for factor VIII to assess vascular density. Immunohistochemistry using specific antibodies was also performed for transforming growth factor-beta, basic-fibroblast growth factor, and vasoendothelial growth factor. Growth factor expression was quantitated by comparing areas of staining (in mm2) with computerized morphometric analysis. RESULTS Mortality was similar in both groups (5/25 versus 8/28; not significant). Group II had significantly greater vascular density than group I (5.65 versus 4.06 vessels/high-power field; p < 0.001), with a peak at 1 week postoperatively (9.12 versus 5.56 vessels/high-power field; p < 0.0001) in both groups. Overall, levels of both transforming growth factor-beta and basic-fibroblast growth factor were significantly higher in the TMR group compared with the control group (0.207 versus 0.141 mm2/mm2, p < 0.05; and 0.125 versus 0.099 mm2/ mm2, p < 0.05). CONCLUSIONS This model of TMR is associated with a significant angiogenic response, which appears to be mediated by the release of certain angiogenic growth factors such as transforming growth factor-beta and basic-fibroblast growth factor. With the long-term patency of laser-created myocardial channels in clinical TMR increasingly in doubt, its mechanism of myocardial revascularization may be similar to that observed in our model.

ALK1 opposes ALK5/Smad3 signaling and expression of extracellular matrix components in human chondrocytes.

Introduction: TGF‐β is a multifunctional regulator of chondrocyte proliferation, differentiation, and extracellular matrix production. Dysregulation of TGF‐β action has been implicated in cartilage diseases such as osteoarthritis. TGF‐β signaling is transduced through a pair of transmembrane serine/threonine kinases, known as the type I (ALK5) and type II receptors. However, recent studies on endothelial cells have identified ALK1 as a second type I TGF‐β receptor and have shown that ALK1 and ALK5 have opposing functions in these cells. Here we examined ALK1 expression and its regulation of TGF‐β signaling and responses in human chondrocytes.

Identification of CD109 as part of the TGF-β receptor system in human keratinocytes

We have previously reported that keratinocytes defective in glycosylphosphatidylinositol (GPI)‐anchor biosynthesis display enhanced TGF‐β responses. These studies implicated the involvement of a 150 kDa GPI‐anchored TGF‐β1 binding protein, r150, in modulating TGF‐β signaling. Here, we sought to determine the molecular identity of r150 by affinity purification and microsequencing. Our results identify r150 as CD109, a novel member of the α2‐macroglob‐ulin (α2M)/complement superfamily, whose function has remained obscure. In addition, we have identified a novel CD109 isoform that occurs in the human placenta but not keratinocytes. Biochemical studies show that r150 contains an internal thioester bond, a defining feature of the α2M/complement family. Loss and gain of function studies demonstrate that CD109 is a component of the TGF‐β receptor system, and a negative modulator of TGF‐β responses in keratinocytes, as implicated for r150. Our data suggest that CD109 can inhibit TGF‐β signaling independently of ligand sequestration and may exert its effect on TGF‐β signaling by direct modulation of receptor activity. Together, our results linking CD109 function to regulation of TGF‐β signaling suggest that CD109 plays a unique role in the regulation of isoform‐specific TGF‐β signaling in keratinocytes.—Finnson, K. W., Tam, B. Y. Y., Liu, K., Marcoux, A., Lepage, P., Roy, S., Bizet, A. A., Philip, A. Identification of CD109 as a TGF‐β1 accessory receptor in human keratinocytes. FASEB J. 20, E780–E795 (2006)

Transforming Growth Factor: β Signaling Is Essential for Limb Regeneration in Axolotls

Axolotls (urodele amphibians) have the unique ability, among vertebrates, to perfectly regenerate many parts of their body including limbs, tail, jaw and spinal cord following injury or amputation. The axolotl limb is the most widely used structure as an experimental model to study tissue regeneration. The process is well characterized, requiring multiple cellular and molecular mechanisms. The preparation phase represents the first part of the regeneration process which includes wound healing, cellular migration, dedifferentiation and proliferation. The redevelopment phase represents the second part when dedifferentiated cells stop proliferating and redifferentiate to give rise to all missing structures. In the axolotl, when a limb is amputated, the missing or wounded part is regenerated perfectly without scar formation between the stump and the regenerated structure. Multiple authors have recently highlighted the similarities between the early phases of mammalian wound healing and urodele limb regeneration. In mammals, one very important family of growth factors implicated in the control of almost all aspects of wound healing is the transforming growth factor-beta family (TGF-β). In the present study, the full length sequence of the axolotl TGF-β1 cDNA was isolated. The spatio-temporal expression pattern of TGF-β1 in regenerating limbs shows that this gene is up-regulated during the preparation phase of regeneration. Our results also demonstrate the presence of multiple components of the TGF-β signaling machinery in axolotl cells. By using a specific pharmacological inhibitor of TGF-β type I receptor, SB-431542, we show that TGF-β signaling is required for axolotl limb regeneration. Treatment of regenerating limbs with SB-431542 reveals that cellular proliferation during limb regeneration as well as the expression of genes directly dependent on TGF-β signaling are down-regulated. These data directly implicate TGF-β signaling in the initiation and control of the regeneration process in axolotls.

Fetal and adult human skin fibroblasts display intrinsic differences in contractile capacity

One of the differences between fetal and adult skin healing is the unique ability of fetal wounds to heal without contracture and scar formation. Studies have shown that the ratio between the three isoforms of TGFβ is different in adult and fetal wounds. Thus, we analyzed the capacity of adult and fetal human skin fibroblasts to contract collagen gels after stimulation with TGFβ isoforms. In control medium, fetal fibroblasts had a contractile capacity similar to that of adult fibroblasts. However, the growth capacity of fetal fibroblasts was completely inhibited, in contrast to adult fibroblasts. When cells were treated with TGFβ, fetal fibroblasts showed an inhibition of their contractile capacity whereas adult fibroblasts further contracted gels. The contractile response was similar for all isoforms of TGFβ although TGFβ3 always had the strongest effect. We considered that the regulation of cell contractile capacity by TGFβ may be dependent on receptor expression for this cytokine, on myofibroblast differentiation of the cells, or in cell links with matrix. Since TGFβ receptor analysis did not show differences in receptor affinity, we studied the expression of α‐smooth muscle (SM) actin, a fibroblast contractile marker and of three integrins, the cell surface receptors specific of the attachment of the fibroblasts with collagen matrix. We observed that the expression of α‐SM actin and α3 and β1 integrin subunits was increased when TGFβ was added to the medium of adult fibroblasts whereas the levels of the α1 and α2 subunits were unchanged. In contrast, fetal fibroblasts treated with TGFβ showed a decrease of α1, α2, and β1 integrin expression but no change in α3 integrin and in α‐SM actin expression. These results indicate that intrinsic differences between fetal and adult fibroblasts might explain their opposite responses to TGFβ stimuli. The variations in their α‐SM actin and integrin expression patterns represent potentially important mechanisms used by fetal fibroblasts to regulate their response to cytokines, and likely contribute to the resultant differences in the quality of wound repair.

The TGF-β co-receptor, CD109, promotes internalization and degradation of TGF-β receptors

Transforming growth factor-β (TGF-β) is implicated in numerous pathological disorders, including cancer and mediates a broad range of biological responses by signaling through the type I and II TGF-β receptors. Internalization of these receptors via the clathrin-coated pits pathway facilitates SMAD-mediated signaling, whereas internalization via the caveolae pathway is associated with receptor degradation. Thus, molecules that modulate receptor endocytosis are likely to play a critical role in regulating TGF-β action. We previously identified CD109, a GPI-anchored protein, as a TGF-β co-receptor and a negative regulator of TGF-β signaling. Here, we demonstrate that CD109 associates with caveolin-1, a major component of the caveolae. Moreover, CD109 increases binding of TGF-β to its receptors and enhances their internalization via the caveolae. In addition, CD109 promotes localization of the TGF-β receptors into the caveolar compartment in the presence of ligand and facilitates TGF-β-receptor degradation. Thus, CD109 regulates TGF-β receptor endocytosis and degradation to inhibit TGF-β signaling. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

Endoglin differentially regulates TGF-β-induced Smad2/3 and Smad1/5 signalling and its expression correlates with extracellular matrix production and cellular differentiation state in human chondrocytes

OBJECTIVE Transforming growth factor-β (TGF-β) plays a critical role in cartilage homeostasis and deregulation of its signalling is implicated in osteoarthritis (OA). TGF-β isoforms signal through a pair of transmembrane serine/threonine kinases known as the type I and type II TGF-β receptors. Endoglin is a TGF-β co-receptor that binds TGF-β with high affinity in the presence of the type II TGF-β receptor. We have previously shown that endoglin is expressed in human chondrocytes and that it forms a complex with the TGF-β signalling receptors. However, the functional significance of endoglin expression in chondrocytes is unknown. Our objective was to determine whether endoglin regulates TGF-β/Smad signalling and extracellular matrix (ECM) production in human chondrocytes and whether its expression varies with chondrocyte differentiation state. METHOD Endoglin function was determined by overexpression or antisense morpholino/siRNA knockdown of endoglin in human chondrocytes and measuring TGF-β-induced Smad phosphorylation, transcriptional activity and ECM production. Alterations in endoglin expression levels were determined during subculture-induced dedifferentiation of human chondrocytes and in normal vs OA cartilage samples. RESULTS Endoglin enhances TGF-β1-induced Smad1/5 phosphorylation and inhibits TGF-β1-induced Smad2 phosphorylation, Smad3-driven transcriptional activity and ECM production in human chondrocytes. In addition, the enhancing effect of endoglin siRNA knockdown on TGF-β1-induced Smad3-driven transcription is reversed by ALK1 overexpression. Furthermore, endoglin levels are increased in chondrocytes following subculture-induced dedifferentiation and in OA cartilage as compared to normal cartilage. CONCLUSION Together, our results suggest that endoglin regulates the balance between TGF-β/ALK1/Smad1/5 and ALK5/Smad2/3 signalling and ECM production in human chondrocytes and that endoglin may represent a marker for chondrocyte phenotype.

CD109‐mediated degradation of TGF‐β receptors and inhibition of TGF‐β responses involve regulation of SMAD7 and Smurf2 localization and function

Transforming growth factor‐β (TGF‐β) is a multifunctional cytokine that regulates a wide variety of cellular processes including proliferation, differentiation, and extracellular matrix deposition. Dysregulation of TGF‐β signaling is associated with several diseases such as cancer and tissue fibrosis. TGF‐β signals through two transmembrane proteins known as the type I (TGFBR1) and type II (TGFBR2) receptors. The levels of these receptors at the cell surface are tightly regulated by several mechanisms, including degradation following recruitment of the E3 ubiquitin ligase SMAD ubiquitination regulatory factor (Smurf) 2 by SMAD7. In addition, TGF‐β co‐receptors can modulate TGF‐β signaling receptor activity in a cell‐specific manner. We have previously identified a novel TGF‐β co‐receptor, CD109, a glycosyl phosphatidylinositol (GPI)‐anchored protein that negatively regulates TGF‐β signaling. Despite CD109s potential relevance as a regulator of TGF‐β action in vivo, the mechanisms by which CD109 regulates TGF‐β signaling are still incompletely understood. Previously, we have shown that CD109 downregulates TGF‐β signaling by promoting TGF‐β receptor localization into the lipid raft/caveolae compartment and by enhancing TGF‐β receptor degradation. Here, we demonstrate that CD109 enhances SMAD7/Smurf2‐mediated degradation of TGFBR1 in a ligand‐dependent manner. Moreover, we show that CD109 regulates the localization and the association of SMAD7/Smurf2 with TGFBR1. Finally, we demonstrate that CD109s inhibitory effect on TGF‐β signaling and responses require SMAD7 expression and Smurf2 ubiquitin ligase activity. Taken together, these results suggest that CD109 is an important regulator of SMAD7/Smurf2‐mediated degradation of TGFBR1. J. Cell. Biochem. 113: 238–246, 2012.

Transforming growth factor-β receptors on human endometrial cells: identification of the type I, II, and III receptors and glycosylphosphatidylinositol anchored TGF-β binding proteins

In the present study, we have characterized the cell surface receptors for transforming growth factor-beta (TGF-beta) on monolayer cultures of stromal cells prepared from human endometrial biopsies, and on a human endometrial epithelial cell line (RL95-2) using affinity cross-link labeling techniques. On the stromal cells, five TGF-beta binding proteins were identified. Analysis of the sensitivity of these proteins to dithiothreitol and phosphatidylinositol-specific phospholipase C, together with results from immunoprecipitations with antibodies against the type II and III TGF-beta receptors, confirmed that three of these binding proteins correspond to the cloned type I, II, and III TGF-beta receptors. The other two binding proteins observed exhibit the characteristics of isoform-specific GPI-anchored TGF-beta binding proteins. On RL95-2 cells, three TGF-beta binding proteins, corresponding to the type I, II, and III TGF-beta receptors, were identified. The receptors which we have characterized on endometrial cells are responsive to physiological concentrations of TGF-beta as demonstrated by the effect of TGF-beta on endometrial cell proliferation. Accordingly, these receptors have the potential to respond to the TGF-beta isoforms which have recently been detected in the endometrium in an autocrine and/or paracrine manner.

Endoglin Is Expressed on Human Chondrocytes and Forms a Heteromeric Complex With Betaglycan in a Ligand and Type II TGFβ Receptor Independent Manner

Previous work has implicated transforming growth factor β (TGFβ) as an essential mediator of cartilage repair and TGFβ signaling as a requirement for the maintenance of articular cartilage in vivo. However, the mechanisms regulating TGFβ action in chondrocytes are poorly understood. Endoglin, an accessory receptor of the TGFβ receptor superfamily, is highly expressed on endothelial cells and has been shown to potently modulate TGFβ responses. It is not known whether chondrocytes express endoglin or whether it modulates TGFβ signaling in these cells. In this study, we show that endoglin is expressed on human chondrocytes at levels comparable with endothelial cells and that it forms higher order complexes with the types I and II TGFβ receptors. More importantly, we show that endoglin forms a heteromeric complex with betaglycan on these cells at endogenous receptor concentrations and ratios. Endoglin complexes with betaglycan in a ligand‐independent and ‐dependent manner as indicated by co‐immunoprecipitation in the absence of TGFβ and after affinity labeling with radiolabeled TGFβ, respectively. Also, the endoglin‐betaglycan association can occur independently of the type II TGFβ receptor. These findings, taken together with the available evidence that endoglin and betaglycan are potent modulators of TGFβ signal transduction, imply that the complex formation between endoglin and betaglycan may be of critical significance in the regulation of TGFβ signaling in chondrocytes.