Novel Paracrine Functions of Smooth Muscle Cells in Supporting Endothelial Regeneration Following Arterial Injury.

RATIONALE: Regeneration of denuded or injured endothelium is an important component of vascular injury response. Cell-cell communication between endothelial cells and smooth muscle cells (SMCs) plays a critical role not only in vascular homeostasis but also in disease. We have previously demonstrated that PKCδ (protein kinase C-delta) regulates multiple components of vascular injury response including apoptosis of SMCs and production of chemokines, thus is an attractive candidate for a role in SMC-endothelial cells communication. OBJECTIVE: To test whether PKCδ-mediated paracrine functions of SMCs influence reendothelialization in rodent models of arterial injury. METHODS AND RESULTS: Femoral artery wire injury was performed in SMC-conditional Prkcd knockout mice, and carotid angioplasty was conducted in rats receiving transient Prkcd knockdown or overexpression. SMC-specific knockout of Prkcd impaired reendothelialization, reflected by a smaller Evans blue-excluding area in the knockout compared with the wild-type controls. A similar impediment to reendothelialization was observed in rats with SMC-specific knockdown of Prkcd. In contrast, SMC-specific gene transfer of Prkcd accelerated reendothelialization. In vitro, medium conditioned by AdPKCδ-infected SMCs increased endothelial wound closure without affecting their proliferation. A polymerase chain reaction-based array analysis identified Cxcl1 and Cxcl7 among others as PKCδ-mediated chemokines produced by SMCs. Mechanistically, we postulated that PKCδ regulates Cxcl7 expression through STAT3 (signal transducer and activator of transcription 3) as knockdown of STAT3 abolished Cxcl7 expression. The role of CXCL7 in SMC-endothelial cells communication was demonstrated by blocking CXCL7 or its receptor CXCR2, both significantly inhibited endothelial wound closure. Furthermore, insertion of a Cxcl7 cDNA in the lentiviral vector that carries a Prkcd shRNA overcame the adverse effects of Prkcd knockdown on reendothelialization. CONCLUSIONS: SMCs promote reendothelialization in a PKCδ-dependent paracrine mechanism, likely through CXCL7-mediated recruitment of endothelial cells from uninjured endothelium.

Left-right axis asymmetry determining human Cryptic gene is transcriptionally repressed by Snail.

BACKGROUND: Establishment of the left-right axis is important for positioning organs asymmetrically in the developing vertebrate-embryo. A number of factors like maternally deposited molecules have emerged essential in initiating the specification of the axis; the downstream events, however, are regulated by signal-transduction and gene-expression changes identifying which remains a crucial challenge. The EGF-CFC family member Cryptic, that functions as a co-receptor for some TGF-beta ligands, is developmentally expressed in higher mammals and mutations in the gene cause loss or change in left-right axis asymmetry. Despite the strong phenotype, no transcriptional-regulator of this gene is known till date. RESULTS: Using promoter-analyses tools, we found strong evidence that the developmentally essential transcription factor Snail binds to the human Cryptic-promoter. We cloned the promoter-region of human Cryptic in a reporter gene and observed decreased Cryptic-promoter activation upon increasing Snail expression. Further, the expression of Cryptic is down-regulated upon exogenous Snail expression, validating the reporter assays and the previously identified role of Snail as a transcriptional repressor. Finally, we demonstrate using gel-shift assay that Snail in nuclear extract of PANC1 cells interacts with the promoter-construct bearing putative Snail binding sites and confirm this finding using chromatin immunoprecipitation assay. CONCLUSIONS: Snail represses the expression of human Cryptic and therefore, might affect the signaling via Nodal that has previously been demonstrated to specify the left-right axis using the EGF-CFC co-receptors.

Necroptosis is associated with Rab27-independent expulsion of extracellular vesicles containing RIPK3 and MLKL.

Extracellular vesicle (EV) secretion is an important mechanism used by cells to release biomolecules. A common necroptosis effector-mixed lineage kinase domain like (MLKL)-was recently found to participate in the biogenesis of small and large EVs independent of its function in necroptosis. The objective of the current study is to gain mechanistic insights into EV biogenesis during necroptosis. Assessing EV number by nanoparticle tracking analysis revealed an increased number of EVs released during necroptosis. To evaluate the nature of such vesicles, we performed a newly adapted, highly sensitive mass spectrometry-based proteomics on EVs released by healthy or necroptotic cells. Compared to EVs released by healthy cells, EVs released during necroptosis contained a markedly higher number of unique proteins. Receptor interacting protein kinase-3 (RIPK3) and MLKL were among the proteins enriched in EVs released during necroptosis. Further, mouse embryonic fibroblasts (MEFs) derived from mice deficient of Rab27a and Rab27b showed diminished basal EV release but responded to necroptosis with enhanced EV biogenesis as the wildtype MEFs. In contrast, necroptosis-associated EVs were sensitive to Ca2+ depletion or lysosomal disruption. Neither treatment affected the RIPK3-mediated MLKL phosphorylation. An unbiased screen using RIPK3 immunoprecipitation-mass spectrometry on necroptotic EVs led to the identification of Rab11b in RIPK3 immune-complexes. Our data suggests that necroptosis switches EV biogenesis from a Rab27a/b dependent mechanism to a lysosomal mediated mechanism.