Angiopoietin-1 regulates microvascular reactivity and protects the microcirculation during acute endothelial dysfunction: role of eNOS and VE-cadherin.

The growth factor angiopoietin-1 (Ang-1) plays an essential role in angiogenesis and vascular homeostasis. Nevertheless, the role of Ang-1 in regulating vascular tone and blood flow is largely unexplored. Endothelial nitric oxide synthase (eNOS) and the junctional protein VE-cadherin are part of the complex signalling cascade initiated by Ang-1 in endothelial cells. In this study, we aimed to investigate the mechanisms underlying acute effects of Ang-1 on microvascular reactivity, permeability and blood flow, and hypothesise that eNOS and VE-cadherin underpin Ang-1 mediated vascular effects that are independent of angiogenesis and proliferation. Myography of isolated microarterioles from male C3H/HeN mice (7-10 weeks) was employed to measure vascular reactivity in vitro. Microcirculatory function in vivo was evaluated by intravital microscopy and Doppler fluximetry in dorsal window chambers. Ang-1 and its stable variant MAT.Ang-1 induced a concentration-dependent vasodilation of arterioles in vitro, which was blocked with nitric oxide (NO) synthesis inhibitor l-NAME. In vivo, MAT.Ang-1 restored to control levels l-NAME induced peripheral vasoconstriction, decreased blood flow and microvascular hyperpermeability. Tissue protein expression of VE-cadherin was reduced by NOS inhibition and restored to control levels by MAT.Ang-1, whilst VE-cadherin phosphorylation was increased by l-NAME and subsequently reduced by MAT.Ang-1 administration. Moreover, MAT.Ang-1 alone did not modulate systemic levels of angiogenetic factors. Our novel findings report that Ang-1 induces arteriolar vasodilation via release of NO, suggesting that Ang-1 is an important regulator of microvascular tone. As MAT.Ang-1 ameliorates detrimental effects on the microcirculation induced by inhibition of NO synthesis and stabilizes the endothelial barrier function through VE-cadherin, we propose that this Ang-1 variant may serve as a novel therapeutic agent to protect the microcirculation against endothelial dysfunction.

Angiopoietin-1 variant reduces LPS-induced microvascular dysfunction in a murine model of sepsis.

INTRODUCTION: Severe sepsis is characterised by intravascular or extravascular infection with microbial agents, systemic inflammation and microcirculatory dysfunction, leading to tissue damage, organ failure and death. The growth factor angiopoietin (Ang-1) has therapeutic potential but recombinant Ang-1 tends to aggregate and has a short half-life in vivo. This study aimed to investigate the acute effects of the more stable Ang-1 variant matrilin-1-angiopoietin-1 (MAT.Ang-1) on the function of the microcirculation in an experimental model of sepsis, and whether any protection by MAT-Ang-1 was associated with modulation of inflammatory cytokines, angiogenic factors or the endothelial nitric oxide synthase (eNOS)-Akt and vascular endothelial (VE)-cadherin pathways. METHODS: Aluminium window chambers were implanted into the dorsal skinfold of male C3H/HeN mice (7 to 10 weeks old) to expose the striated muscle microcirculation. Endotoxemia was induced by intraperitoneal injection of lipopolysaccharide (LPS, 1 mg/kg at 0 and 19 hours). MAT.Ang-1 was administered intravenously 20 hours after the onset of sepsis. Microcirculatory function was evaluated by intravital microscopy and Doppler fluximetry. RESULTS: Endotoxemia resulted in macromolecular leak, which was ameliorated by MAT.Ang-1 post-treatment. LPS induced a dramatic reduction in tissue perfusion, which was improved by MAT.Ang-1. Proteome profiler array analysis of skeletal muscle also demonstrated increased inflammatory and reduced angiogenic factors during endotoxemia. MAT.Ang-1 post-treatment reduced the level of IL-1ß but did not significantly induce the expression of angiogenic factors. MAT.Ang-1 alone did not induce leak or increase angiogenic factors but did reduce vascular endothelial growth factor expression in controls. CONCLUSION: Administration of MAT.Ang-1 after the onset of sepsis protects the microcirculation from endotoxemia-induced vascular dysfunction through reducing inflammation but without pro-angiogenic actions, thus representing a novel, potential pharmacotherapeutic agent for the treatment of sepsis.

Protein disulphide isomerase family members show distinct substrate specificity: P5 is targeted to BiP client proteins.

At least 17 members of the protein disulphide isomerase (PDI) family of oxidoreductases are present in the endoplasmic reticulum (ER) of mammalian cells. They are thought to catalyse disulphide formation to aid folding or to regulate protein function; however, little is known about their individual functions. Here, we show that some proteins that enter the ER are clients for single oxidoreductases, whereas others are clients for several PDI-like enzymes. We previously identified potential substrates for ERp57, and here identify substrates for ERp18 and ERp46. In addition, we analysed the specificity of substrates towards PDI, ERp72, ERp57, ERp46, ERp18 and P5. Strikingly, ERp18 shows specificity towards a component of the complement cascade, pentraxin-related protein PTX3, whereas ERp46 has specificity towards peroxiredoxin-4, a thioredoxin peroxidase. By contrast, most PDI family members react with Ero1alpha. Moreover, P5 forms a non-covalent complex with immunoglobulin heavy chain binding protein (BiP) and shows specificity towards BiP client proteins. These findings highlight cooperation between BiP and P5, and demonstrate that individual PDI family members recognise specific substrate proteins.

Sequence-specific recognition of collagen triple helices by the collagen-specific molecular chaperone HSP47.

HSP47 is a molecular chaperone that plays an unknown role during the assembly and transport of procollagen. Our previous studies showed that, unlike most chaperones, HSP47 interacts with a correctly folded substrate. We suggested that HSP47 either stabilizes the correctly folded collagen helix from heat denaturation or prevents lateral aggregation prior to its transport from the endoplasmic reticulum. In this study we have addressed the role of triple helix stability in the binding of HSP47 to procollagen by expressing procollagen molecules with differing thermal stabilities and analyzing their ability to interact with HSP47 within the endoplasmic reticulum. Our results show that HSP47 interacts with thermostable procollagen molecules, suggesting that helix stabilization is not the primary function of HSP47 and that the interaction of HSP47 with procollagen depends upon the presence of a minimum of one Gly-X-Arg triplet within the triple helical domain. Interestingly, procollagen chains containing high proportions of stabilizing triplets formed triple helices and interacted with HSP47 even in the absence of proline hydroxylation, demonstrating that recognition does not depend upon this modification. Our results support the view that HSP47 functions early in the secretory pathway by preventing the lateral aggregation of procollagen chains.