Alexander G. Minchenko

Oxidized Phospholipids Stimulate Angiogenesis Via Autocrine Mechanisms, Implicating a Novel Role for Lipid Oxidation in the Evolution of Atherosclerotic Lesions

Angiogenesis is a common feature observed in advanced atherosclerotic lesions. We hypothesized that oxidized phospholipids (OxPLs), which accumulate in atherosclerotic vessels can stimulate angiogenesis. We found that oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) stimulated the formation of sprouts from endothelial cell spheroids and promoted growth of capillaries into Matrigel plugs in mice. OxPLs stimulated expression of vascular endothelial growth factor (VEGF) in vivo and in several normal and tumor cell types in vitro. In addition, OxPAPC upregulated cyclooxygenase (COX)-2 and interleukin (IL)-8. COX-2 inhibitors, as well as blocking antibodies to IL-8 suppressed activation of sprouting by OxPAPC. We conclude that OxPAPC stimulates angiogenesis via autocrine mechanisms involving VEGF, IL-8, and COX-2–generated prostanoids. Our data suggest that accumulation of OxPLs may contribute to increased growth of blood capillaries in advanced lesions, thus leading to progression and destabilization of atherosclerotic plaques.

Diabetes-induced overexpression of endothelin-1 and endothelin receptors in the rat renal cortex is mediated via poly(ADP-ribose) polymerase activation

We hypothesize that poly (ADP‐ribosyl)ation, that is, poly (ADP‐ribose) polymerase (PARP)‐dependent transfer of ADP‐ribose moieties from NAD to nuclear proteins, plays a role in diabetic nephropathy. We evaluated whether PARP activation is present and whether two unrelated PARP inhibitors, 3‐aminobenzamide (ABA) and 1,5‐isoquinolinediol (ISO), counteract overexpression of endothelin‐1 (ET‐1) and ET receptors in the renal cortex in short‐term diabetes. The studies were performed in control rats and streptozotocin‐diabetic rats treated with/without ABA or ISO (30 and 3 mg*kg−1*day−1, intraperitoneally, for 2 weeks after 2 weeks of diabetes). Poly (ADP‐ribose) immunoreactivity was increased in tubuli, but not glomeruli, of diabetic rats and this increase was corrected by ISO, whereas ABA had a weaker effect. ET‐1 concentration (ELISA) was increased in diabetic rats, and this elevation was blunted by ISO. ET‐1, ET(A), and ET(B) mRNA (ribonuclease protection assay), but not ET‐3 mRNA (RT/PCR), abundance was increased in diabetic rats, and three variables were, at least, partially corrected by ISO. ABA produced a trend towards normalization of ET‐1 concentration and ET‐1, ET(A), and ET(B) mRNA abundance, but the differences with untreated diabetic group were not significant. Poly(ADP‐ribosyl)ation is involved in diabetes‐induced renal overexpression of ET‐1 and ET receptors. PARP inhibitors could provide a novel therapeutic approach for diabetic complications including nephropathy, and other diseases that involve the endothelin system.

Effect of Recombinant Soluble P-Selectin Glycoprotein Ligand-1 on Leukocyte-Endothelium Interaction In Vivo Role in Rat Traumatic Shock

-Traumatic shock induces profound pathophysiological alterations and initiates inflammatory reactions in many tissues, thus resulting in acute multiple organ damage (eg, intestine, pancreas, and liver). In the rat, Noble-Collip drum trauma increases P-selectin expression on the vascular endothelium as a result of loss of endothelium-derived NO. Here we postulated that blockade of the earliest steps in leukocyte adhesion (ie, leukocyte rolling) via administration of a recombinant soluble form of P-selectin glycoprotein ligand-1 (PSGL-1; the recombinant soluble form is rsPSGL.Ig) would attenuate selectin-mediated events observed in the rat during traumatic shock. Using intravital microscopy of the rat mesenteric microvasculature, we found that intravenous infusion of rsPSGL.Ig significantly inhibited leukocyte-endothelium interaction (ie, leukocyte rolling, adherence, and transmigration) induced by traumatic shock as well as by activation of the microvascular endothelium with 50 micromol/L NG-nitro-L-arginine methyl ester. Immunohistochemical detection of P-selectin on the mesenteric venular endothelial surface demonstrated that rsPSGL.Ig functionally neutralizes effects of P-selectin on the endothelial cell surface rather than attenuating P-selectin expression. Systemic administration of rsPSGL.Ig to traumatized rats prolonged survival time and survival rate, significantly attenuating ileal myeloperoxidase activity and significantly preserving mesenteric endothelial function. Furthermore, PSGL-1 mRNA levels were significantly increased in the blood of traumatized rats and were reduced after systemic administration of rsPSGL.Ig. Thus, soluble recombinant forms of PSGL-1 are able to ameliorate acute shock states by suppressing selectin-mediated leukocyte-endothelium interaction at both the functional and molecular levels.

P-selectin is up-regulated in vital organs during murine traumatic shock.

Murine traumatic shock is associated with increased adherence of neutrophils to the vascular endothelium resulting in neutrophil infiltration and tissue damage. We examined the effects of trauma on the expression of the adhesion molecule P‐selectin in several vital organs (i.e., heart, lungs, liver, kidneys, and small intestine) 2 h after induction of Noble‐Collip drum trauma in anesthetized rats. Total RNA was extracted from these organs and P‐selectin mRNA was quantified by RNase protection assays. P‐selectin mRNA was significantly increased over control nontraumatized, anesthetized rats in all vital organs (P<0.05 or less), with the largest increase occurring in the lung (P<0.01). Immunohistochemical analysis showed increased expression of P‐selectin protein in postcapillary venules of all vital organs after trauma. To further investigate the possible mechanisms of increased P‐selectin mRNA transcription promoter activity during trauma, we quantified binding of proteins from nuclear extracts to the κB site (−218GGGGGTGACCC−207) of the P‐selectin gene by electrophoretic mobility shift assay. We confirmed the results of NF‐κB binding by demonstrating increases in p50 and p52, as well as decreases in IκB in cytoplasmic and nuclear extracts from the lungs of trauma rats by Western blotting. Increased activity of the transcription factor, nuclear factor κB (NF‐κB), occurred in all vital organs of the trauma rats compared to sham‐operated controls. Our findings suggest that severe trauma results in up‐regulation of P‐selectin at the transcriptional level, which is partly controlled by an NF‐κB‐responsive element in the region of the P‐selectin promoter. This increased activation of NF‐κB binding may contribute to the widespread increases in P‐selectin expression observed in several vital organs 2 h after trauma, which in turn may play a key role in the pathogenesis of traumatic shock.—Armstead, V. E., Minchenko, A. G., Campbell, B., Lefer, A. M. P‐selectin is up‐regulated in vital organs during murine traumatic shock. FASEB J. 11, 1271–1279 (1997)

Tissue Factor Expression in Vital Organs during Murine Traumatic Shock Role of Transcription Factors AP-1 and NF-κB

BACKGROUND Tissue factor (TF) is a cell-surface glycoprotein responsible for initiating the extrinsic pathway of coagulation that has been shown to have a role in the pathophysiology of sepsis and reperfusion injury. The purpose of this study was to investigate TF expression in vital organs and to determine possible regulatory mechanisms of TF expression in the lung during traumatic shock in rats. METHODS Noble-Collip drum trauma was induced in anesthetized Sprague-Dawley rats. Anesthetized rats without trauma served as controls. TF activity was measured in plasma and lung tissue. TF messenger RNA (mRNA) was measured in the lung, liver, and small intestine using ribonuclease protection assays. Electromobility shift assays were used to quantify binding of nuclear extracts from lung to TF-specific consensus domains for transcription factors NF-kappaB and AP-1. RESULTS TF activity in plasma increased up to 14-fold and +232% in the lung (P < 0.001 for plasma and lung) 2 h after trauma. TF mRNA level was significantly increased in the lungs (P < 0.01), small intestine (P < 0.01), and liver (P < 0.05) 1 h after trauma compared to sham-operated control rats. TF mRNA expression continued to increase in the lungs and the liver (both, P < 0.001) 2 h after trauma TF sequence-specific complex binding to AP-1 and NF-kappaB domains was enhanced in the lungs of trauma rats (+395%, P < 0.001 and +168%, P < 0.001, respectively). CONCLUSIONS These results suggest that TF may play an important role in the pathophysiology of severe trauma and that regulatory elements AP-1 and NF-kappaB may be involved in the regulation of TF mRNA expression in traumatic shock.

Endothelin-1, Endothelin Receptors and ecNOS Gene Transcription in Vital Organs During Traumatic Shock in Rats

Endothelin-1 (ET-1) is a vasoconstrictor peptide that may play an important role in the pathophysiology of severe trauma. We examined ET-1 gene expression in vital organs (i.e., heart, lungs, kidneys, liver and small intestine) during murine traumatic shock using ribonuclease protection assays. Our data show that ET-1 mRNA was significantly increased in the lungs two hours after trauma when compared with control anesthetized rats. There was also a significant increase in ET-1 transcripts occurring in the kidneys, heart and liver. During these experimental conditions, we also observed statistically significant increased endothelin type B (ET(B)) receptor mRNA expression in the lung, heart, liver, kidney and small intestine. Expression of endothelial constitutive nitric oxide synthase (ecNOS) gene, which is functionally coupled to ET(B) receptor, also was increased in vital organs during traumatic shock. Endothelin type A (ET(A)) receptor gene expression was slightly decreased in the lung, liver and small intestine. These results suggest that ET-1 and ET(B) mRNA expression are mainly increased in the lung and other vital organs and may play a functional role in the pathophysiology of murine traumatic shock.

Pulmonary tissue factor mRNA expression during murine traumatic shock: effect of P-selectin blockade.

Tissue factor (TF) is the primary cellular initiator of the coagulation protease cascade and serves as a cell surface receptor and a specific cofactor for plasma factors VII/VIIa. Because there is evidence that TF is regulated by a P-selectin dependent gene, we examined TF mRNA expression in the lungs during murine traumatic shock in the presence and absence of recombinant soluble P-selectin glycoprotein ligand-1 (rsPSGL.Ig) by using ribonuclease protection assays. Moreover, we studied the level of TF mRNA expression in mice with their P-selectin gene deleted (P-selectin -/-). Our data show that TF mRNA was significantly increased (+143%; P < 0.001) in the lungs 2 h after trauma compared with control rats subjected to sham trauma, which exhibited reduced TF mRNA expression (-34%; P < 0.001) after systemic administration of rsPSGL.Ig. The expression of TF mRNA was also significantly decreased (-29%; P < 0.05) in the lungs of P-selectin -/- mice compared with wild-type control C57B16 mice. The present results provide evidence for a P-selectin-dependent mechanism that enhances TF gene expression in traumatic shock. The major support for this mechanism is that either blockade of P-selectin by rsPSGL.Ig or deletion of the P-selectin gene leads to significant decreases in TF mRNA expression in the lung. These results are consistent with the concept that TF interacting with P-selectin may play a significant role in the pathophysiology of trauma.