Reversible renal glomerular dysfunction in guinea pigs exposed to glutaraldehyde-polymerized cell-free hemoglobin.

Chemically modified hemoglobin (Hb)-based oxygen carriers are promising oxygen replacement therapeutics however their potential renal effects are not fully understood. Using a guinea pig exchange transfusion model, we examined the effects of glutaraldehyde-polymerized bovine hemoglobin (HbG) on the permeability and integrity of the glomerular filtration barrier (GFB), which is comprised of podocytes, fenestrated endothelium, and the glomerular basement membrane. HbG induced marked proteinuria characterized in part by the loss of high molecular weight proteins, including albumin, immunoglobulin, and transferrin, at 4 and 12 h post-infusion that resolved by 72 h.

CD36 mediates H2O2-induced calcium influx in lung microvascular endothelial cells.

Elevated levels of reactive oxygen species and intracellular Ca play a key role in endothelial barrier dysfunction in acute lung injury. We previously showed that HO-induced increases in intracellular calcium concentrations ([Ca]) in lung microvascular endothelial cells (LMVECs) involve the membrane Ca channel, transient receptor potential vanilloid-4 (TRPV4) and that inhibiting this channel attenuated HO-induced barrier disruption in vitro. We also showed that phosphorylation of TRPV4 by the Src family kinase, Fyn, contributes to HO-induced Ca influx in LMVEC.

Transcriptional Suppression of Renal Antioxidant Enzyme Systems in Guinea Pigs Exposed to Polymerized Cell-Free Hemoglobin.

Hemoglobin-based oxygen carriers (HBOCs) are being developed as oxygen and plasma volume-expanding therapeutics though their potential to promote oxidative tissue injury has raised safety concerns. Using a guinea pig exchange transfusion model, we examined the effects of polymerized bovine hemoglobin (HbG) on the transcriptional regulation, activity, and expression of the renal antioxidant enzymes; superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). HbG infusion downregulated the mRNA levels for genes encoding SOD isoforms 1-3, GPx1, GPx3, GPx4, and CAT.

Nitric oxide synthase promotes distension-induced tracheal venular leukocyte adherence.

The process of leukocyte recruitment to the airways in real time has not been extensively studied, yet airway inflammation persists as a major contributor to lung pathology. We showed previously in vivo, that neutrophils are recruited acutely to the large airways after periods of airway distension imposed by the application of positive end-expiratory pressure (PEEP). Given extensive literature implicating products of nitric oxide synthase (NOS) in lung injury after ventilatory over-distension, we questioned whether similar mechanisms exist in airway post-capillary venules.

Phosphodiesterase 2A is a major negative regulator of iNOS expression in lipopolysaccharide-treated mouse alveolar macrophages.

PDE2A is a dual-function PDE that is stimulated by cGMP to hydrolyze cAMP preferentially. In a two-hit model of ALI, we found previously that PDE2A decreased lung cAMP, up-regulated lung iNOS, and exacerbated ALI. Recent data suggest that macrophage iNOS expression contributes to ALI but later, promotes lung-injury resolution.

Protein kinase G increases antioxidant function in lung microvascular endothelial cells by inhibiting the c-Abl tyrosine kinase.

Oxidant injury contributes to acute lung injury (ALI). We previously reported that activation of protein kinase GI (PKGI) posttranscriptionally increased the key antioxidant enzymes catalase and glutathione peroxidase 1 (Gpx-1) and attenuated oxidant-induced cytotoxicity in mouse lung microvascular endothelial cells (MLMVEC). The present studies tested the hypothesis that the antioxidant effect of PKGI is mediated via inhibition of the c-Abl tyrosine kinase.

CD36 and Fyn kinase mediate malaria-induced lung endothelial barrier dysfunction in mice infected with Plasmodium berghei.

Severe malaria can trigger acute lung injury characterized by pulmonary edema resulting from increased endothelial permeability. However, the mechanism through which lung fluid conductance is altered during malaria remains unclear. To define the role that the scavenger receptor CD36 may play in mediating this response, C57BL/6J (WT) and CD36-/- mice were infected with P.

Knockdown of lung phosphodiesterase 2A attenuates alveolar inflammation and protein leak in a two-hit mouse model of acute lung injury.

Phosphodiesterase 2A (PDE2A) is stimulated by cGMP to hydrolyze cAMP, a potent endothelial barrier-protective molecule. We previously found that lung PDE2A contributed to a mouse model of ventilator-induced lung injury (VILI). The purpose of the present study was to determine the contribution of PDE2A in a two-hit mouse model of 1-day intratracheal (IT) LPS followed by 4 h of 20 ml/kg tidal volume ventilation.

Evolutionarily conserved, growth plate zone-specific regulation of the matrilin-1 promoter: L-Sox5/Sox6 and Nfi factors bound near TATA finely tune activation by Sox9.

To help uncover the mechanisms underlying the staggered expression of cartilage-specific genes in the growth plate, we dissected the transcriptional mechanisms driving expression of the matrilin-1 gene (Matn1). We show that a unique assembly of evolutionarily conserved cis-acting elements in the Matn1 proximal promoter restricts expression to the proliferative and prehypertrophic zones of the growth plate. These elements functionally interact with distal elements and likewise are capable of restricting the domain of activity of a pancartilaginous Col2a1 enhancer.

cGMP increases antioxidant function and attenuates oxidant cell death in mouse lung microvascular endothelial cells by a protein kinase G-dependent mechanism.

Increasing evidence suggests that endothelial cytotoxicity from reactive oxygen species (ROS) contributes to the pathogenesis of acute lung injury. Treatments designed to increase intracellular cGMP attenuate ROS-mediated apoptosis and necrosis in several cell types, but the mechanisms are not understood, and the effect of cGMP on pulmonary endothelial cell death remains controversial. In the current study, increasing intracellular cGMP by either 8pCPT-cGMP (50 microM) or atrial natriuretic peptide (10 nM) significantly attenuated cell death in H(2)O(2)-challenged mouse lung microvascular (MLMVEC) monolayers.

Soluble guanylyl cyclase contributes to ventilator-induced lung injury in mice.

High tidal volume (HV(T)) ventilation causes pulmonary endothelial barrier dysfunction. HV(T) ventilation also increases lung nitric oxide (NO) and cGMP. NO contributes to HV(T) lung injury, but the role of cGMP is unknown.

Role of vasodilator-stimulated phosphoprotein in cGMP-mediated protection of human pulmonary artery endothelial barrier function.

Increased pulmonary endothelial cGMP was shown to prevent endothelial barrier dysfunction through activation of protein kinase G (PKG(I)). Vasodilator-stimulated phosphoprotein (VASP) has been hypothesized to mediate PKG(I) barrier protection because VASP is a cytoskeletal phosphorylation target of PKG(I) expressed in cell-cell junctions. Unphosphorylated VASP was proposed to increase paracellular permeability through actin polymerization and stress fiber bundling, a process inhibited by PKG(I)-mediated phosphorylation of Ser(157) and Ser(239).

Highly conserved proximal promoter element harbouring paired Sox9-binding sites contributes to the tissue- and developmental stage-specific activity of the matrilin-1 gene.

The matrilin-1 gene has the unique feature that it is expressed in chondrocytes in a developmental stage-specific manner. Previously, we found that the chicken matrilin-1 long promoter with or without the intronic enhancer and the short promoter with the intronic enhancer restricted the transgene expression to the columnar proliferative chondroblasts and prehypertrophic chondrocytes of growth-plate cartilage in transgenic mice. To study whether the short promoter shared by these transgenes harbours cartilage-specific control elements, we generated transgenic mice expressing the LacZ reporter gene under the control of the matrilin-1 promoter between -338 and +67.

Functional analysis of the regulatory regions of the matrilin-1 gene in transgenic mice reveals modular arrangement of tissue-specific control elements.

Matrilin-1 is a non-collagenous protein, which functions in the organization of the extracellular matrix by forming collagen-dependent and -independent filamentous networks. It is secreted primarily by chondrocytes in a characteristic spatial, temporal and developmental stage-specific pattern during skeletogenesis. As a first step to define the tissue- and site-specific regulatory regions of the chicken matrilin-1 gene in vivo, we generated transgenic mice harboring various promoter and intronic fragments fused to the LacZ reporter gene.