Proteoglycan 4 modulates osteogenic smooth muscle cell differentiation during vascular remodeling and intimal calcification

Calcification is a prominent feature of late-stage atherosclerosis, but the mechanisms driving this process are unclear. Using a biobank of carotid endarterectomies, we recently showed that Proteoglycan 4 (PRG4) is a key molecular signature of calcified plaques, expressed in smooth muscle cell (SMC) rich regions. Here, we aimed to unravel the PRG4 role in vascular remodeling and intimal calcification. PRG4 expression in human carotid endarterectomies correlated with calcification assessed by preoperative computed tomographies. PRG4 localized to SMCs in early intimal thickening, while in advanced lesions it was found in the extracellular matrix, surrounding macro-calcifications. In experimental models, Prg4 was upregulated in SMCs from partially ligated ApoE(-/-) mice and rat carotid intimal hyperplasia, correlating with osteogenic markers and TGFb1. Furthermore, PRG4 was enriched in cells positive for chondrogenic marker SOX9 and around plaque calcifications in ApoE(-/-) mice on warfarin. In vitro, PRG4 was induced in SMCs by IFNg, TGFb1 and calcifying medium, while SMC markers were repressed under calcifying conditions. Silencing experiments showed that PRG4 expression was driven by transcription factors SMAD3 and SOX9. Functionally, the addition of recombinant human PRG4 increased ectopic SMC calcification, while arresting cell migration and proliferation. Mechanistically, it suppressed endogenous PRG4, SMAD3 and SOX9, and restored SMC markers' expression. PRG4 modulates SMC function and osteogenic phenotype during intimal remodeling and macro-calcification in response to TGFb1 signaling, SMAD3 and SOX9 activation. The effects of PRG4 on SMC phenotype and calcification suggest its role in atherosclerotic plaque stability, warranting further investigations.Vascular Surger

Cytoskeletal protein degradation in brain death donor kidneys associates with adverse posttransplant outcomes

In brain death, cerebral injury contributes to systemic biological dysregulation, causing significant cellular stress in donor kidneys adversely impacting the quality of grafts. Here, we hypothesized that donation after brain death (DBD) kidneys undergo proteolytic processes that may deem grafts susceptible to posttransplant dysfunction. Using mass spectrometry and immunoblotting, we mapped degradation profiles of cytoskeletal proteins in deceased and living donor kidney biopsies. We found that key cytoskeletal proteins in DBD kidneys were proteolytically cleaved, generating peptide fragments, predominantly in grafts with suboptimal posttransplant function. Interestingly, alpha-actinin-4 and talin-1 proteolytic fragments were detected in brain death but not in circulatory death or living donor kidneys with similar donor characteristics. As talin-1 is a specific proteolytic target of calpain-1, we investigated a potential trigger of calpain activation and talin-1 degradation using human ex vivo precision-cut kidney slices and in vitro podocytes. Notably, we showed that activation of calpain-1 by transforming growth factor-beta generated proteolytic fragments of talin-1 that matched the degradation fragments detected in DBD preimplantation kidneys, also causing dysregulation of the actin cytoskeleton in human podocytes; events that were reversed by calpain-1 inhibition. Our data provide initial evidence that brain death donor kidneys are more susceptible to cytoskeletal protein degradation. Correlation to posttransplant outcomes may be established by future studies

Níveis de proteína bruta na dieta após o desmame e desempenho em leitões Crude protein levels in the post-weaning diets and performance in piglets

Foram comparados três níveis de proteína, com leitões desmamados aos 29 dias de idade, no período de 0 a 15 dias após o desmame. Os tratamentos foram os seguintes: T1- dieta testemunha, com 20% de proteína bruta (PB); T2 - dieta com 18% PB; T3 - dieta com 16% PB. As dietas eram isolisínicas com 1,15% de lisina. Durante a fase de aleitamento foi fornecida uma dieta pré-inicial a todos os leitões, a partir do sétimo dia de vida. De 16 a 36 dias após o desmame, foi fornecida uma dieta com 18% de PB a todos os leitões. A redução do nível de PB da dieta para 18% ou 16% não afetou (P>0,10) o desempenho dos leitões em nenhum dos períodos estudados e reduziu (P<0,01) a incidência e severidade da diarréia. Concluiu-se que a redução do nível de PB e da proporção do farelo de soja na dieta de leitões desmamados aos 29 dias de idade, acompanhada de suplementação com lisina, por 15 dias após o desmame, pode proporcionar redução na incidência e severidade da diarréia, sem afetar o desempenho.<br>Three crude protein levels were compared with weaning piglets (29 days of age), in the period from 0 to 15 days post-weaning. The treatments compared were: T1- control diet, 20% crude protein (CP); T2 - 18% CP diet; T3 - 16% CP diet. Diets were isolisinic, containing 1,15% total lysine. During the weanling phase a pre-starter diet was fed to all piglets, from seventh day of age. An 18% CP diet was fed to all piglets, from 16 to 36 days post-weaning. The reduction of CP level in the diet to 18% or 16% did not affect (P>0,10) the performance of piglets in any of the studied periods and it decreased (P<0,01) the incidence and severity of diarrhea. It was concluded that the reduction in the crude protein level and in the proportion of soybean meal on the diet for piglets weaned at 29 days, fortified with lysine, fed for 15 days post-weaning, decrease the incidence and severity of diarrhea, and not decrease the performance