Notch3 expression in capillary pericytes predicts worse graft outcome in human renal grafts with antibody-mediated rejection.

Microvasculature consisting of endothelial cells and pericytes is the main site of injury during antibody-mediated rejection (ABMR) of renal grafts. Little is known about the mechanisms of activation of pericytes in this pathology. We have found recently that activation of Notch3, a mediator of vascular smooth muscle cell proliferation and dedifferentiation, promotes renal inflammation and fibrosis and aggravates progression of renal disease. Therefore, we studied the pericyte expression of Notch3 in 49 non-selected renal graft biopsies (32 for clinical cause, 17 for graft surveillance). We analysed its relationship with patients' clinical and morphological data, and compared with the expression of partial endothelial mesenchymal transition (pEndMT) markers, known to reflect endothelial activation during ABMR. Notch3 was de novo expressed in pericytes of grafts with ABMR, and was significantly correlated with the microcirculation inflammation scores of peritubular capillaritis and glomerulitis and with the expression of pEndMT markers. Notch3 expression was also associated with graft dysfunction and proteinuria at the time of biopsy and in the long term. Multivariate analysis confirmed pericyte expression of Notch3 as an independent risk factor predicting graft loss. These data suggest that Notch3 is activated in the pericytes of renal grafts with ABMR and is associated with poor graft outcome.

Notch3 orchestrates epithelial and inflammatory responses to promote acute kidney injury.

Acute kidney injury is a major risk factor for subsequent chronic renal and/or cardiovascular complications. Previous studies have shown that Notch3 was de novo expressed in the injured renal epithelium in the early phases of chronic kidney disease. Here we examined whether Notch3 is involved in the inflammatory response and the epithelial cell damage that typifies ischemic kidneys using Notch3 knockout mice and mice with short-term activated Notch3 signaling (N3ICD) in renal epithelial cells. After ischemia/reperfusion, N3ICD mice showed exacerbated infiltration of inflammatory cells and severe tubular damage compared to control mice. Inversely, Notch3 knockout mice were protected against ischemia/reperfusion injury. Renal macrophages derived from Notch3 knockout mice failed to activate proinflammatory cytokines. Chromatin immunoprecipitation analysis of the Notch3 promoter identified NF-κB as the principal inducer of Notch3 in ischemia/reperfusion. Thus, Notch3 induced by NF-κB in the injured epithelium sustains a proinflammatory environment attracting activated macrophages to the site of injury leading to a rapid deterioration of renal function and structure. Hence, targeting Notch3 may provide a novel therapeutic strategy against ischemia/reperfusion and acute kidney injury by preservation of epithelial structure and disruption of proinflammatory signaling.

Markers of Endothelial-to-Mesenchymal Transition: Evidence for Antibody-Endothelium Interaction during Antibody-Mediated Rejection in Kidney Recipients.

Antibody-mediated rejection (ABMR) is a leading cause of allograft loss. Treatment efficacy depends on accurate diagnosis at an early stage. However, sensitive and reliable markers of antibody-endothelium interaction during ABMR are not available for routine use. Using immunohistochemistry, we retrospectively studied the diagnostic value of three markers of endothelial-to-mesenchymal transition (EndMT), fascin1, vimentin, and heat shock protein 47, for ABMR in 53 renal transplant biopsy specimens, including 20 ABMR specimens, 24 cell-mediated rejection specimens, and nine normal grafts. We validated our results in an independent set of 74 unselected biopsy specimens. Endothelial cells of the peritubular capillaries in grafts with ABMR expressed fascin1, vimentin, and heat shock protein 47 strongly, whereas those from normal renal grafts did not. The level of EndMT marker expression was significantly associated with current ABMR criteria, including capillaritis, glomerulitis, peritubular capillary C4d deposition, and donor-specific antibodies. These markers allowed us to identify C4d-negative ABMR and to predict late occurrence of disease. EndMT markers were more specific than capillaritis for the diagnosis and prognosis of ABMR and predicted late (up to 4 years after biopsy) renal graft dysfunction and proteinuria. In the independent set of 74 renal graft biopsy specimens, the EndMT markers for the diagnosis of ABMR had a sensitivity of 100% and a specificity of 85%. Fascin1 expression in peritubular capillaries was also induced in a rat model of ABMR. In conclusion, EndMT markers are a sensitive and reliable diagnostic tool for detecting endothelial activation during ABMR and predicting late loss of allograft function.

Mechanistic insight from murine models of Netherton syndrome.

Protease regulation plays a crucial role in skin homeostasis and inflammation as revealed by the identification of loss-of-function mutations in SPINK5 (serine protease inhibitor of Kazal type 5) in Netherton sydrome (NS). SPINK5 encodes LEKTI (lympho-epithelial Kazal type related inhibitor), a multidomain serine protease inhibitor expressed in all stratified epithelia. Our laboratory has developed a number of murine models which have been instrumental in dissecting the pathogenesis of NS. This minireview discusses the major findings of these models and emphasizes the role of protease regulation, especially kallikrein-related peptidases in NS.

Activation of Notch3 in Glomeruli Promotes the Development of Rapidly Progressive Renal Disease.

Notch3 expression is found in the glomerular podocytes of patients with lupus nephritis or focal segmental GN but not in normal kidneys. Here, we show that activation of the Notch3 receptor in the glomeruli is a turning point inducing phenotypic changes in podocytes promoting renal inflammation and fibrosis and leading to disease progression. In a model of rapidly progressive GN, Notch3 expression was induced by several-fold in podocytes concurrently with disease progression. By contrast, mice lacking Notch3 expression were protected because they exhibited less proteinuria, uremia, and inflammatory infiltration. Podocyte outgrowth from glomeruli isolated from wild-type mice during the early phase of the disease was higher than outgrowth from glomeruli of mice lacking Notch3. In vitro studies confirmed that podocytes expressing active Notch3 reorganize their cytoskeleton toward a proliferative/migratory and inflammatory phenotype. We then administered antisense oligodeoxynucleotides targeting Notch3 or scramble control oligodeoxynucleotides in wild-type mice concomitant to disease induction. Both groups developed chronic renal disease, but mice injected with Notch3 antisense had lower values of plasma urea and proteinuria and inflammatory infiltration. The improvement of renal function was accompanied by fewer deposits of fibrin within the glomeruli and by decreased peritubular inflammation. Finally, abnormal Notch3 staining was observed in biopsy samples of patients with crescentic GN. These results demonstrate that abnormal activation of Notch3 may be involved in the progression of renal disease by promoting migratory and proinflammatory pathways. Inhibiting Notch3 activation could be a novel, promising approach to treat GN.

Expression of sarco (endo) plasmic reticulum calcium ATPase (SERCA) system in normal mouse cardiovascular tissues, heart failure and atherosclerosis.

UNLABELLED: The sarco(endo)plasmic reticulum Ca(2+)ATPases (SERCA) system, a key regulator of calcium cycling and signaling, is composed of several isoforms. We aimed to characterize the expression of SERCA isoforms in mouse cardiovascular tissues and their modulation in cardiovascular pathologies (heart failure and/or atherosclerosis). Five isoforms (SERCA2a, 2b, 3a, 3b and 3c) were detected in the mouse heart and thoracic aorta. Absolute mRNA quantification revealed SERCA2a as the dominant isoform in the heart (~99%). Both SERCA2 isoforms co-localized in cardiomyocytes (CM) longitudinal sarcoplasmic reticulum (SR), SERCA3b was located at the junctional SR. In the aorta, SERCA2a accounted for ~91% of total SERCA and SERCA2b for ~5%. Among SERCA3, SERCA3b was the most expressed (~3.3%), mainly found in vascular smooth muscle cells (VSMC), along with SERCA2a and 2b. In failing CM, SERCA2a was down-regulated by 2-fold and re-localized from longitudinal to junctional SR. A strong down-regulation of SERCA2a was also observed in atherosclerotic vessels containing mainly synthetic VSMCs. The proportion of both SERCA2b and SERCA3b increased to 9.5% and 8.3%, respectively. IN CONCLUSION: 1) SERCA2a is the major isoform in both cardiac and vascular myocytes; 2) the expression of SERCA2a mRNA is ~30 fold higher in the heart compared to vascular tissues; and 3) nearly half the amount of SERCA2a mRNA is measured in both failing cardiomyocytes and synthetic VSMCs compared to healthy tissues, with a relocation of SERCA2a in failing cardiomyocytes. Thus, SERCA2a is the principal regulator of excitation-contraction coupling in both CMs and contractile VSMCs.

The Notch pathway attenuates interleukin 1ß (IL1ß)-mediated induction of adenylyl cyclase 8 (AC8) expression during vascular smooth muscle cell (VSMC) trans-differentiation.

Vascular smooth muscle cell (VSMC) trans-differentiation, or their switch from a contractile/quiescent to a secretory/inflammatory/migratory state, is known to play an important role in pathological vascular remodeling including atherosclerosis and postangioplasty restenosis. Several reports have established the Notch pathway as tightly regulating VSMC response to various stress factors through growth, migration, apoptosis, and de-differentiation. More recently, we showed that alterations of the Notch pathway also govern VSMC acquisition of the inflammatory state, one of the major events accelerating atherosclerosis. We also evidenced that the inflammatory context of atherosclerosis triggers a de novo expression of adenylyl cyclase isoform 8 (AC8), associated with the properties developed by trans-differentiated VSMCs. As an initial approach to understanding the regulation of AC8 expression, we examined the role of the Notch pathway. Here we show that inhibiting the Notch pathway enhances the effect of IL1ß on AC8 expression, amplifies its deleterious effects on the VSMC trans-differentiated phenotype, and decreases Notch target genes Hrt1 and Hrt3. Conversely, Notch activation resulted in blocking AC8 expression and up-regulated Hrt1 and Hrt3 expression. Furthermore, overexpressing Hrt1 and Hrt3 significantly decreased IL1ß-induced AC8 expression. In agreement with these in vitro findings, the in vivo rat carotid balloon-injury model of restenosis evidenced that AC8 de novo expression coincided with down-regulation of the Notch3 pathway. These results, demonstrating that the Notch pathway attenuates IL1ß-mediated AC8 up-regulation in trans-differentiated VSMCs, suggest that AC8 expression, besides being induced by the proinflammatory cytokine IL1ß, is also dependent on down-regulation of the Notch pathway occurring in an inflammatory context.

Implication of adenylyl cyclase 8 in pathological smooth muscle cell migration occurring in rat and human vascular remodelling.

Recently, we discovered on primary cell cultures that adenylyl cyclase type 8 (AC8) was involved in the transition of rat vascular smooth muscle cells (VSMCs) to an inflammatory phenotype. Here we demonstrate, in human vessels displaying early or advanced atherosclerotic lesions, that: (a) only intimal VSMCs strongly express AC8; and (b) very few AC8-positive VSMCs were detected in the medial layer, either in atherosclerotic or healthy arteries. Furthermore, over-expressing AC8 in primary rat VSMC cultures triggered the recolonization of a wounded zone and similar results were obtained in the presence of mitomycin, a potent inhibitor of proliferation. This phenomenon was prevented by silencing AC8. Indeed, in IL-1 beta-treated cells, AC8 silencing halted migration and decreased the matrix-metalloproteinases 2/9 secretion, known to be involved in VSMC migration. In vivo, we showed: (a) a pronounced up-regulation of AC8 expression in highly migrating VSMCs of the injured rat carotid artery; (b) an undetectable AC8 labelling in re-endothelized vessels where neo-intimal thickening had stopped. From our data, we conclude that AC8 expression appears closely linked to the properties developed by VSMCs in atherosclerosis and post-angioplasty neo-intima formation leading to restenosis. In addition, it reinforces the idea that VSMC responses to their cell environment greatly depend on the AC isoforms expressed and attributes a new role for AC8 in these pathological vascular processes.

ReGeneraTing Agents (rgta®) technology combined with antibiotics improves outcomes for infections in the upper limb.

A matrix therapy agent marketed as CACIPLIQ20® showed marked improvement in the healing rate of hand infections, including functional recovery. It can be used at both earlier and later stages to promote faster healing and prevent an adverse outcome.

TSLP-activated dendritic cells induce human T follicular helper cell differentiation through OX40-ligand.

T follicular helper cells (Tfh) are important regulators of humoral responses. Human Tfh polarization pathways have been thus far associated with Th1 and Th17 polarization pathways. How human Tfh cells differentiate in Th2-skewed environments is unknown. We show that thymic stromal lymphopoietin (TSLP)-activated dendritic cells (DCs) promote human Tfh differentiation from naive CD4 T cells. We identified a novel population, distinct from Th2 cells, expressing IL-21 and TNF, suggestive of inflammatory cells. TSLP-induced T cells expressed CXCR5, CXCL13, ICOS, PD1, BCL6, BTLA, and SAP, among other Tfh markers. Functionally, TSLP-DC-polarized T cells induced IgE secretion by memory B cells, and this depended on IL-4Rα. TSLP-activated DCs stimulated circulating memory Tfh cells to produce IL-21 and CXCL13. Mechanistically, TSLP-induced Tfh differentiation depended on OX40-ligand, but not on ICOS-ligand. Our results delineate a pathway of human Tfh differentiation in Th2 environments.