L’apoptose des cellules endothéliales (CE) représente un évènement initial dans le développement de plusieurs pathologies fibrotiques telles que le rejet chronique d’allogreffe et la sclérose systémique. Nous avons démontré que les médiateurs issus des CE apoptotiques entraîne la différenciation myofibroblastique et la résistance à l’apoptose, deux mécanismes centraux à la fibrogénèse. L’activation de PI3K (phospatidylinositol-3 kinase) caractérise ces deux mécanismes. Un fragment C-terminal du perlécan (LG3) produit par les CE apoptotiques inhibe l’apoptose des fibroblastes. Les objectifs de ce travail étaient de : 1. définir les récepteurs et la signalisation impliqués dans la réponse anti-apoptotique et 2. caractériser les médiateurs fibrogéniques responsables de la différenciation myofibroblastique. En ce qui a trait à la réponse anti-apoptotique, l’inhibition des intégrines 21 ou des kinases de la famille Src (SFK) chez les fibroblastes prévient la résistance à l’apoptose et la phosphorylation d’Akt normalement induites par le milieu conditionné par des CE apoptotiques (SSC) ou le LG3. Ces résultats suggèrent que le LG3 produit par les CE apoptotiques initie un état de résistance à l’apoptose chez les fibroblastes par des voies α2β1integrines/SFK/PI3K dépendantes. Le LG3 n’induit cependant pas la différenciation myofibroblastique. Nous avons donc caractérisé le milieu SSC de façon à identifier les médiateurs responsables de la différenciation myofibroblastique. Les milieux conditionnés par des CE apoptotiques et non-apoptotiques (respectivement SSC et SSC-ZVAD) ont été analysés comparativement par chromatographie liquide bi-dimensionnelle, immunobuvardage et spectrométrie de masse. Le connective tissue growth factor (CTGF) est le seul facteur fibrogénique connu augmenté dans le milieu SSC. L’inhibition de la caspase-3 chez les CE prévient la relâche de CTGF. Au niveau du fibroblaste, l’inhibition de SFK ou de Pyk2 (proline-rich tyrosine kinase-2) prévient la différenciation myofibroblastique induite par le SSC ou le CTGF in vitro. L’anticorps neutralisant contre le TGF- (Transforming growth factor beta) n’est pas en mesure de bloquer la différenciation myofibroblastique induite par le SSC ou le CTGF. Des injections quotidiennes sous-cutanées de SSC chez la souris C3H pour 3 semaines entraîne une augmentation de l’épaisseur de la peau et des niveaux protéiques d’SMA, de vimentine et de collagène I. Cette réponse fibrogénique est réduite chez les souris qui ont reçu le SSC-ZVAD ou le SSC immunodéplété de son CTGF. Ces résultats apportent de nouvelles issues mécanistiques au niveau de la réponse fibrogénique activée par la mort des CE. L’activation des caspases chez les CE apoptotiques entraîne la production de LG3 et de CTGF qui, à leur tour, activent des voies de signalisation pro-fibrotiques SFK/PI3K dépendantes chez les fibroblastes, et ce indépendamment du TGF-. / Apoptosis of endothelial cells (EC) is an early event in various fibrotic diseases including chronic allograft vasculopathy and systemic sclerosis. We showed previously that mediators released by apoptotic EC activate myofibroblast differentiation and resistance to apoptosis, two mechanisms pivotal to fibrogenesis. PI3K (phospatidylinositol-3 kinase) activation was found to be central to these two mechanisms. A C-terminal fragment of perlecan (LG3) produced by apoptotic EC was found to inhibit apoptosis of fibroblasts. The aims of the present project were : 1. to define the receptors and pathways implicated in this anti-apoptotic response and 2. to characterize the fibrogenic mediators implicated in myofibroblast differentiation. Concerning the anti-apoptotic response, the inhibition of 21 integrin activity in fibroblasts exposed to either medium conditioned by apoptotic EC (SSC) or LG3 prevented resistance to apoptosis and was associated with decreased levels of Akt phosphorylation. Neutralizing Src family kinases (SFK) activity in fibroblasts produced the same effects. These results suggest that LG3 produced by apoptotic EC initiate a state of resistance to apoptosis in fibroblasts via an α2β1integrin/SFK/PI3K dependent pathway. LG3 did not induce myofibroblast differentiation. We went on to identify which mediators present in SSC are implicated in myofibroblast differentiation. Media conditioned by apoptotic and non-apoptotic EC (respectively SSC and SSC-ZVAD) were analyzed comparatively by 2-dimension liquid chromatography, western blotting and mass spectrometry. Connective tissue growth factor (CTGF) was the only known fibrogenic factor increased in SSC. Caspase-3 silencing of EC demonstrated that CTGF is released by apoptotic EC downstream of caspase-3 activation. In fibroblasts, blocking the activation of SFK or silencing the proline-rich tyrosine kinase 2 (Pyk2) blocked myofibroblast differentiation triggered by either SSC or recombinant CTGF in vitro. Exposure to a pan-transforming growth factor (TGF-β) neutralizing antibody failed to attenuate myofibroblast differentiation in fibroblasts exposed to either SSC or CTGF. Subcutaneous injection of mouse SSC to C3H mice daily for three weeks led to increased skin thickness, increased protein levels of αSMA, vimentin and collagen I. This fibrogenic response was blunted in mice injected with either SSC-ZVAD or SSC immunodepleted of CTGF. These results bring new mechanistic insights into the fibrogenic pathways activated by EC death. Caspase activation in apoptotic EC triggers the production of LG3 and CTGF which in turn activate SFK/PI3K dependant pathways in fibroblasts thus activating a TGF-β-independent fibrogenic response.

Indiana University-Purdue University Indianapolis (IUPUI) / Pyk2: Potential Regulator of Post-Menopausal Bone Loss H.W. LARGURA1,2*, P. ELENISTE2, S. HUANG2, S. LIU1, M. ALLEN3, A. BRUZZANITI2. 1Indiana University School of Dentistry Department Orthodontics and Oral Facial Development, 2Indiana University School of Dentistry Department of Oral Biology, 3Indiana University School of Medicine Department of Anatomy and Cell Biology, Indianapolis, Indiana, USA Osteoporosis is a pathologic condition of bone, commonly found in post-menopausal women, which occurs from an imbalance between bone formation and resorption. Following menopause, the bone resorbing activity of osteoclasts exceeds bone formation by osteoblasts, resulting in decreased trabecular and cortical bone and a subsequent decrease in bone mass. Reduced bone mass increases the risk of pathologic fracture of bones. Due to adverse effects associated with current treatment protocols for bone loss, alternative treatment modalities with reduced adverse effects are needed. Estrogen plays a role in maintaining balance in the bone remodeling cycle by controlling remodeling activation, osteoblast and osteoclast numbers, and their respective effectiveness in formation and resorption. With declining estrogen levels, this elegantly balanced interaction is altered and bone resorption exceeds bone formation, resulting in bone loss and increased bone fragility. Pyk2 is a protein tyrosine kinase that plays an important role in regulating bone resorption by osteoclasts, as well as osteoblast proliferation and differentiation. Deletion of the Pyk2 gene in mice leads to an increase in bone mass, in part due to dysfunctional osteoclast and osteoblast activity. In this study, we examined the role of Pyk2 in the effects of estrogen on bone mass. We used wild type (WT) and Pyk2 knock-out (KO) mice that had been ovariectomized (OVX) and treated with or without estrogen (E2)-releasing pellets. Control mice included sham OVX surgery receiving placebo pellet. We found that deletion of Pyk2 conferred increased bone mass in sham, OVX and OVX+E2 mice. In addition, Pyk2 KO mice supplemented with 17estradiol exhibited a marked increase in bone volume/trabecular volume, trabecular number, and trabecular thickness, but not cortical bone parameters compared to WT mice. Results of this study provide evidence for the role of Pyk2 in the effects of estrogen on bone mass. Understanding the role of Pyk2 in bone could lead to the development of new pharmaceutical targets for the treatment of bone loss associated with osteoporosis.

Indiana University-Purdue University Indianapolis (IUPUI) / Introduction: Regenerative endodontic procedures are an effective treatment option for immature teeth with infected necrotic pulps to allow for healing and potential continued root development, yet challenges to ideal treatment outcomes remain. Consistent development of root length and width of dentin remains a challenge, as does development of the pulp-dentin complex. Previous in vitro studies have assessed the role of different growth factors and bioactive molecules in combination with scaffolds to potentially facilitate continued development of the pulp-dentin complex using dental pulp stem cells (DPSCs). The proline-rich tyrosine kinase 2 (Pyk2) is linked with osteoblast activity and the regulation of bone mass. Further, the Pyk2 inhibitor PF-4618433 (PF-46) has been shown in previous studies to enhance osteoblast activity and mineral deposition in vitro. However, whether Pyk2 targeting promotes the osteogenic differentiation of DPSCs remains unknown. Objective: The purpose of this study was to investigate the effect of a Pyk2 inhibitor, PF-46, on the proliferation, differentiation, and mineralization of human DPSCs. Materials and Methods: Human DPSCs were cultured in 24-well plates with α-MEM with 10% FBS, and containing 0 μM (vehicle control) or 0.1 μM, 0.3 μM, or 0.6 μM PF-46. Fresh media and treatments were replaced every 2-3 days. After 1 day incubation, cytotoxic effects were evaluated by using an MTS proliferation assay. After 4 days of treatment, direct cell counting was performed. To induce osteogenic differentiation, ascorbic acid and β-glycerol phosphate were added to the culture media and the DPSCs were cultured with PF-46 for 14 days. Then, an alkaline phosphatase (ALP) assay and mineral deposition assay were performed. Differences between treatment groups were analyzed by a one-way ANOVA followed by pair-wise tests conducted using Tukey’s multiple comparisons procedure with a 5% significance level. Results: The 0.6 μM PF-46 group had a significantly higher cell count, ALP activity and mineral deposition when compared to 0 μM PF-46. The 0.1 and 0.3 μM PF-46 groups also had significantly higher ALP activity compared to the 0 μM PF-46 group after 14 days of incubation. There was a general trend of increased differentiation and mineral deposition as the concentration of PF-46 increased from 0.1 μM to 0.6 μM. Conclusion: There was a general concentration-dependent increase in cell count, differentiation, and mineral deposition by human DPSCs as the concentration of PF-46 increased from 0 μM up to 0.6 μM, with the highest activity observed with 0.6 μM PF-46. Although further research is needed, these results suggest that strategies that target Pyk2 may potentially be used to improve the osteogenic differentiation of DPSCs to aid endodontic regeneration.

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