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Spinal cord injury (SCI) leads to rapid destruction of neuronal tissue, resulting in devastating motor and sensory deficits. This is exacerbated by damage to spinal cord blood vessels and loss of vascular integrity. Thus, approaches that protect existing blood vessels or stimulate the growth of new blood vessels might present a novel approach to minimize loss or promote regeneration of spinal cord tissue following SCI. In light of the remarkable power of chronic mild hypoxia (CMH) to stimulate vascular remodeling in the brain, the goal of this study was to examine how CMH (8% O for up to 7 days) affects blood vessel remodeling in the spinal cord. We found that CMH promoted the following: (1) endothelial proliferation and increased vascularity as a result of angiogenesis and arteriogenesis, (2) increased vascular expression of the angiogenic extracellular matrix protein fibronectin as well as concomitant increases in endothelial expression of the fibronectin receptor α5β1 integrin, (3) strongly upregulated endothelial expression of the tight junction proteins claudin-5, ZO-1 and occludin and (4) astrocyte activation. Of note, the vascular remodeling changes induced by CMH were more extensive in white matter. Interestingly, hypoxic-induced vascular remodeling in spinal cord blood vessels was markedly attenuated in mice lacking endothelial α5 integrin expression (α5-EC-KO mice). Taken together, these studies demonstrate the considerable remodeling potential of spinal cord blood vessels and highlight an important angiogenic role for the α5β1 integrin in promoting endothelial proliferation. They also imply that stimulation of the α5β1 integrin or controlled use of mild hypoxia might provide new approaches for promoting angiogenesis and improving vascular integrity in spinal cord blood vessels.

BackgroundExtracellular matrix (ECM) proteins play critical functions regulating vascular formation and function. Laminin is a major component of the vascular basal lamina, and transgenic mice deficient in astrocyte or pericyte laminin show defective blood-brain barrier (BBB) integrity, indicating an important instructive role for laminin in cerebral blood vessels. As previous work shows that in the normal brain, vascular expression of the laminin receptor α6β4 integrin is predominantly restricted to arterioles, but induced on all vessels during neuroinflammation, it is important to define the role of this integrin in the maintenance of BBB integrity.Methodsα6β4 integrin expression was analyzed using dual immunofluorescence (dual-IF) of brain sections taken from the mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). To investigate the role of endothelial α6β4 integrin, transgenic mice lacking β4 integrin in endothelial cells (β4-EC-KO) and wild-type (WT) littermates were subject to EAE, and clinical score and various neuropathological parameters were examined by immunofluorescence. In addition, β4 integrin null brain endothelial cells (BECs) were examined in culture for expression of tight junction proteins using immunocytochemistry and flow cytometry.ResultsCerebrovascular expression of β4 integrin was markedly upregulated during EAE progression, such that by the acute stage of EAE (day 21), the vast majority of blood vessels expressed β4 integrin. In the EAE model, while the β4-EC-KO mice showed the same time of disease onset as the WT littermates, they developed significantly worse clinical disease over time, resulting in increased clinical score at the peak of disease and maintained elevated thereafter. Consistent with this, the β4-EC-KO mice showed enhanced levels of leukocyte infiltration and BBB breakdown and also displayed increased loss of the endothelial tight junction proteins claudin-5 and ZO-1. Under pro-inflammatory conditions, primary cultures of β4KO BECs also showed increased loss of claudin-5 and ZO-1 expression.ConclusionsTaken together, our data suggest that α6β4 integrin upregulation is an inducible protective mechanism that stabilizes the BBB during neuroinflammatory conditions.

Remote preconditioning is a unique phenomenon in which brief episodes of ischemia and reperfusion to remote organ protect the target organ against sustained ischemia-reperfusion (I/R)-induced injury. Protective effects of remote renal preconditioning (RRPC) are well established in heart, but their mechanisms still remain to be elucidated. So, the present study was designed to investigate the possible role of oxygen-sensing hypoxia inducible factor-prolyl 4-hydroxylases (HIF-P4Hs) in RRPC-induced cardioprotection in rats. Remote renal preconditioning was performed by four episodes of 5 min renal artery occlusion and reperfusion. Isolated rat hearts were perfused on Langendorff apparatus and were subjected to global ischemia for 30 min followed by 120 min reperfusion. The levels of lactate dehydrogenase (LDH) and creatine kinase (CK) were measured in coronary effluent to assess the degree of myocardial injury. Extent of myocardial infarct size and coronary flow rate was also measured. Ethyl 3,4-dihydroxybenzoate (EDHB) and alpha-ketoglutarate (alpha-KG) were employed as HIF-P4Hs inhibitor and activator, respectively. Diethyldithiocarbamic acid (DDCA) was employed as NFkB inhibitor. Remote renal preconditioning prevented I/R-induced myocardial injury and produced cardioprotective effects. Pharmacological preconditioning with EDHB (100 mg kg(-1) i.p.) mimicked the cardioprotective effects of RRPC. However, alpha-KG (200 mg kg(-1) i.p.) and DDCA (150 mg kg(-1) i.p.) abolished cardioprotective effects of RRPC and EDHB. So, it may be concluded that inhibition of HIF-P4H has a key role in RRPC-induced cardioprotection. Further, remote preconditioning-induced HIF-P4H inhibition may have triggered a transduction pathway involving activation of NFkB.

Multidimensional single cell-analyses of T cells have fueled the debate about either extensive plasticity or “mixed” priming of T helper cell subsets in vivo. Here, we developed an experimental framework to probe the idea that the site of priming in the systemic immune compartment is a determinant of T helper cell-induced immunopathology in remote organs. By site-specific in vivo labeling of antigen-specific T cells in inguinal (i) or gut draining mesenteric (m) lymph nodes, we show that i-T cells and m-T cells isolated from the inflamed central nervous system in a model of multiple sclerosis are distinct. i-T cells were Cxcr6 + and m-T cells expressed P2rx7. Notably, m-T cells infiltrated white matter while i-T cells were also recruited to grey matter. Therefore, we propose that the definition of T helper cell subsets by their site of priming might guide an advanced understanding of T helper cell biology in health and disease.

It has been recently reported that release of erythropoietin could mediate the cardioprotective effects of remote renal preconditioning. However, the mechanism of erythropoietin-mediated cardioprotection in remote preconditioning is still unexplored. Therefore, the present study was designed to investigate the possible signal transduction pathway of erythropoietin-mediated cardioprotection in remote preconditioning in rats. Remote renal preconditioning was performed by four episodes of 5 min renal artery occlusion followed by 5 min reperfusion. Isolated rat hearts were perfused on Langendorff apparatus and were subjected to global ischemia for 30 min followed by 120 min reperfusion. The levels of lactate dehydrogenase (LDH) and creatine kinase (CK) were measured in coronary effluent to assess the degree of myocardial injury. Extent of myocardial infarct size and coronary flow rate was also measured. Remote renal preconditioning and erythropoietin preconditioning (5,000 IUkg(-1), i.p.) attenuated ischemia-reperfusion-induced myocardial injury and produced cardioprotective effects. However, administration of diethyldithiocarbamic acid (150 mg kg(-1) i.p.), a selective NFkB inhibitor, and glibenclamide (5 mg kg(-1) i.p.), a selective K(ATP) channel blocker, attenuated cardioprotective effects of remote preconditioning and erythropoietin preconditioning. However, administration of minoxidil (1 mg kg(-1) i.v.), a selective K(ATP) channel opener, restored the attenuated cardioprotective effects of remote preconditioning and erythropoietin preconditioning in diethyldithiocarbamic acid pretreated rats. These results suggest that K(ATP) channel is a downstream mediator of NFkB activation in remote preconditioning and erythropoietin preconditioning. Therefore, it may be concluded that erythropoietin preconditioning and remote renal preconditioning trigger similar signaling mechanisms for cardioprotection, i.e., NFkB activation followed by opening of K(ATP) channels.

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