RESUMO
BACKGROUND: We examined how prekallikrein (PK) activation on human microvascular endothelial cells (HMVECs) is regulated by the ambient concentration of C1 inhibitor (C1INH) and prolylcarboxypeptidase (PRCP). OBJECTIVE: We sought to examine the specificity of PK activation on HMVECs by PRCP and the role of C1INH to regulate it, high-molecular-weight kininogen (HK) cleavage, and bradykinin (BK) liberation. METHODS: Investigations were performed on cultured HMVECs. Immunofluorescence, enzymatic activity assays, immunoblots, small interfering RNA knockdowns, and cell transfections were used to perform these studies. RESULTS: Cultured HMVECs constitutively coexpressed PK, HK, C1INH, and PRCP. PK activation on HMVECs was modulated by the ambient C1INH concentration. In the absence of C1INH, forming PKa on HMVECs cleaved 120-kDa HK completely to a 65-kDa H-chain and a 46-kDa L-chain in 60 minutes. In the presence of 2 µM C1INH, only 50% of the HK became cleaved. C1INH concentrations (0.0-2.5 µM) decreased but did not abolish BK liberated from HK by activated PK. Factor XII did not activate when incubated with HMVECs alone for 1 hour. However, if incubated in the presence of HK and PK, factor XII became activated. The specificity of PK activation on HMVECs by PRCP was shown by several inhibitors to each enzyme. Furthermore, PRCP small interfering RNA knockdowns magnified C1INH inhibitory activity on PK activation, and PRCP transfections reduced C1INH inhibition at any given concentration. CONCLUSIONS: These combined studies indicated that on HMVECs, PK activation and HK cleavage to liberate BK were modulated by the local concentrations of C1INH and PRCP.
Assuntos
Fator XII , Pré-Calicreína , Humanos , Células Endoteliais , Bradicinina/farmacologia , Cininogênio de Alto Peso Molecular , RNA Interferente Pequeno/genéticaRESUMO
A previously hemostatically asymptomatic patient with common variable hypogammaglobulinemia was given everolimus to prevent growth of her liver. Within several months, the patient developed a severe bleeding disorder. The bleeding was due to fibrin polymerization defect that upon sequencing was shown to be dysfibrinogenemia Krakow III. Elimination of the mTor inhibitor ameliorated the clinical bleeding state.
RESUMO
Reciprocity of inflammation, oxidative stress and neovascularization is emerging as an important mechanism underlying numerous processes from tissue healing and remodelling to cancer progression. Whereas the mechanism of hypoxia-driven angiogenesis is well understood, the link between inflammation-induced oxidation and de novo blood vessel growth remains obscure. Here we show that the end products of lipid oxidation, ω-(2-carboxyethyl)pyrrole (CEP) and other related pyrroles, are generated during inflammation and wound healing and accumulate at high levels in ageing tissues in mice and in highly vascularized tumours in both murine and human melanoma. The molecular patterns of carboxyalkylpyrroles are recognized by Toll-like receptor 2 (TLR2), but not TLR4 or scavenger receptors on endothelial cells, leading to an angiogenic response that is independent of vascular endothelial growth factor. CEP promoted angiogenesis in hindlimb ischaemia and wound healing models through MyD88-dependent TLR2 signalling. Neutralization of endogenous carboxyalkylpyrroles impaired wound healing and tissue revascularization and diminished tumour angiogenesis. Both TLR2 and MyD88 are required for CEP-induced stimulation of Rac1 and endothelial migration. Taken together, these findings establish a new function of TLR2 as a sensor of oxidation-associated molecular patterns, providing a key link connecting inflammation, oxidative stress, innate immunity and angiogenesis.