John D. Edwards

Functional and morphological assessment of rat aorta stored in University of Wisconsin and Eurocollins solutions

University of Wisconsin (UW) and Eurocollins (EC) solutions are widely used for preservation of organs before transplantation. However, effect of storage solutions on vascular interface for transplant success is not known. In this study, we have used rat aorta as a model and assessed the effects of cold storage in UW and EC solutions on smooth muscle and endothelial function and the morphology. Smooth muscle and endothelial functions of the rat aorta were assessed using in vitro isometric tension measurement. Morphologic studies were done with scanning and transmission electron microscopy. No significant difference in contractile response to either norepinephrine (NE) or potassium chloride was observed between control aorta and aorta stored in UW solution for 1 hr or 24 hr. In contrast, sensitivity, but not the reactivity to NE and KCl, was increased in aorta stored in EC solution for 1 hr. If the tissues were stored in EC solution for 24 hr, both sensitivity and reactivity to NE and KCl were significantly reduced. Relaxatory response to acetylcholine, in endothelium-intact vessels were reduced in aortas stored in EC solution, but not in UW solution. The magnitude of relaxations observed in tissues stored in the EC solution for 24 hr was less than in tissues stored for 1 hr. Sodium nitroprusside elicited similar relaxatory response in en-dothelium-denuded control tissue and in tissues stored in UW and EC solution. Electron microscopy data revealed marked swelling of the cell, loss of mitochondria and other intracellular organelles, and striking calcium deposits after preservation of the vessels in EC for 1 or 24 hr. In aorta stored in UW solution for 24 hr, endothelial and smooth muscle cells were intact, with moderate-size vacuoles in the cytoplasm. These results suggest that the UW solution is more suitable than EC solution for short-term preoperative storage of blood vessels.

Insulin like growth factor-1 activates nuclear factor-κB and increases transcription of the intercellular adhesion molecule-1 gene in endothelial cells

A critical early event in the pathogenesis of occlusive vascular disease is the adhesion of monocytes to endothelial cells. The authors have previously reported that insulin-like growth factor-1 increases monocyte-endothelial cell adhesion and increases the expression of intercellular adhesion molecule-1. In this study, it is hypothesized that the upregulation of intercellular adhesion molecule-1 expression after treatment with insulin-like growth factor-1 is caused by an increase in the transcription of intercellular adhesion molecule-1 in endothelial cells, and that this transcription is regulated, at least in part, by activation of nuclear factor-kappaB. Adherence cell assays were performed using insulin-like growth factor-1 treated human umbilical vein endothelial cells and human monocytes. To determine the role of nuclear factor-kappaB, Western blotting using the anti-p65 (activated portion of nuclear factor-kappaB) was performed on cell lysate of human umbilical vein endothelial cells treated with insulin-like growth factor-1. RT-PCR was performed on RNA extracted from insulin-like growth factor-1-treated human umbilical vein endothelial cells. Intercellular adhesion molecule-1 antibody attenuated the increase in monocyte-endothelial cell adhesion of endothelial cells exposed to insulin-like growth factor-1. We observed an increase in expression of the activated nuclear factor-kappaB p65 protein in response to insulin-like growth factor-1 treatment. Peak increase occurred at 30 min. This effect was sensitive to pretreatment of human umbilical vein endothelial cells with the insulin-like growth factor-1 receptor antibody. Human umbilical vein endothelial cells treated with insulin-like growth factor-1 for 2 and 4 h revealed a significant increase in intercellular adhesion molecule-1 mRNA as compared with untreated human umbilical vein endothelial cells. Tumor necrosis factor-alpha produced a larger increase in intercellular adhesion molecule-1 mRNA expression. These results suggest that insulin-like growth factor-1 enhances intercellular adhesion molecule-1 transcription and activates nuclear factor-kappaB in endothelial cells. The intracellular pathways that increase cell adhesion molecule expression may provide a common link to understanding the monocyte-endothelial cell adhesion that occurs in the early stages of atherosclerosis and restenosis.

Cell adhesion molecules and insulin-like growth factor-1 in vascular disease☆☆☆★

PURPOSE Recent advances in the understanding of the biologic mechanisms of vascular diseases suggest that multifactorial stimulation of the endothelial cell and its subsequent adhesion to leukocytes is a prerequisite to the formation of atherosclerotic and restenotic lesions. As leukocyte-endothelial cell interaction is coordinated by a variety of cell adhesion molecules (CAMs), we hypothesized that the expression of certain CAMs is up-regulated in the vasculature of patients who have peripheral vascular disease. In addition, we proposed that insulin-like growth factor-1 (IGF-1) increases monocyte-endothelial adhesion by means of upregulation of these CAMs. METHODS Using immunohistochemical techniques, the expression of intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, and P-selectin was examined in human vascular disease specimens. Normal aortas obtained from the organ retrieval system were studied as control specimens. Adhesion studies between human umbilical vein endothelial cells (HUVECs) incubated with IGF-1 and purified human blood monocytes labeled with 51chromium were completed. Western blotting and flow cytometry were performed to show CAM expression on IGF-1-treated HUVECs. RESULTS Of the CAMs, ICAM-1, P-selectin, and E-selectin were distinctly increased in diseased specimens when compared with control specimens (p < 0.05). Adhesion studies showed an increase in monocyte-endothelial cell adhesion of as much as 40% to 45% (p < 0.01) over baseline, with peak adherence occurring 4 hours after treatment with IGF-1. IGF-1 increased adherence in a dose- and time-dependent manner. The threshold concentration of IGF-1 that induced increased adhesion was 20 ng/ml, with a maximum effect occurring at 150 ng/ml. This increased adhesion was attenuated by pretreatment with IGF-I receptor antibody, as well as with genistein and herbimycin-A, which are potent and selective tyrosine kinase inhibitors. Increased adhesion correlated with an increase in the expression of CAMs on the surface of the HUVECs. An additive effect on adhesion was observed between IGF-1 and tumor necrosis factor-alpha (TNF-alpha) and endothelin-1 (ET-1). Finally, immunohistochemical analysis of human vascular disease specimens revealed an increased expression of IGF-1 receptors as compared with control specimens (p < 0.05). CONCLUSIONS These results suggest that IGF-1 may be important in the pathogenesis of peripheral vascular disease by increasing endothelial cell-monocyte adhesion by means of an increase in the expression of ICAM-1 and VCAM-1.

G-proteins in rat blood vessels—II. Assessment of functional involvement☆

1. In this study, we compared G-protein-mediated contractile responses of rat aorta and mesenteric artery rings induced by the alpha-adrenoceptor agonist, norepinephrine (NE) and by the direct G-protein activator, sodium fluoride, using various probes. 2. Activator of the stimulatory G-protein (Gs), cholera toxin (CT), attenuated the sensitivity and maximum contractile response of both aorta and mesenteric artery to NE and sodium fluoride. The effect of the toxin on the NE-sensitivity was greater in mesenteric artery. 3. Pretreatment of tissues with the inhibitor of Gi-protein, pertussis toxin (PT), reduced the sensitivity as well as maximum contraction of both the aorta and mesenteric artery to sodium fluoride, and of the mesenteric artery to NE. PT attenuated only the sensitivity but not the maximum contraction of the aorta to NE. The inhibitory effect of PT on sensitivity to NE or sodium fluoride was greater in the aorta. 4. NE and sodium fluoride-induced contractions were reduced by the sulfhydryl G-protein inhibitor, N-ethylmaleimide (NEM) in both blood vessels. NEM produced greater inhibitory effect on the sensitivity of the aorta to both contractile agents. 5. These data demonstrate that CT, PT and NEM-sensitive G-proteins are involved in NE- and sodium fluoride-induced contractile responses of the rat aorta and mesenteric artery. The differential effects of the G-protein probes indicate that certain variations in G-protein-mediated contractile responses exist among the two blood vessels, suggesting that G-protein involvement in functional responses may vary with the type of blood vessel investigated.

Short-term preservation of autogenous vein grafts: Effectiveness of University of Wisconsin solution☆

BACKGROUND Suboptimal preservation of autologous veins in storage solutions causes endothelial cell damage that can contribute to graft failure. The purpose of this study was to compare the effects of short-term storage of veins in autologous whole blood (AWB), 0.9% normal saline solution (NS), and University of Wisconsin solution (UWs) on vein structure and function after grafting. METHODS Autogenous jugular and femoral veins were atraumatically harvested from mongrel dogs. One vein segment was immediately implanted to serve as a control, and the other segments were stored for 45 minutes in AWB, NS, or UWs. The veins were implanted as reversed interposition graft in the carotid or femoral arteries. After 6 weeks light and scanning electron microscopy and isometric tension studies were performed on explanted vein grafts. RESULTS Morphologic studies revealed an intact endothelium that stained positively for factor VIII. Intimal thickness was similar between controls (48 +/- 12 microns) and veins stored in UWs (53 +/- 8 microns) (p = not significant), but it was significantly increased in veins stored in AWB (151 +/- 29 microns) and NS (149 +/- 18 microns) (p < 0.05). Sensitivity and maximum contraction to norepinephrine were not altered in veins preserved in UWs (6.0 +/- 0.1 mumol/L and 0.19 +/- 0.02 gm/mm2) but were significantly reduced (p < 0.05) in those stored in AWB (7.2 +/- 0.1 mumol/L and 0.08 +/- 0.02 gm/mm2) and NS (7.0 +/- 0.3 mumol/L and 0.09 +/- 0.02 gm/mm2) compared with controls (5.9 +/- 0.2 mumol/L and 0.20 +/- 0.02 gm/mm2). The sensitivity and maximum relaxation to acetylcholine and sodium nitroprusside of veins preserved in AWB, NS, and UWs were similar to controls (p = not significant). CONCLUSIONS Vein storage in UWs preserves smooth muscle cell function compared with veins stored in NS or AWB. Therefore UWs is a more suitable medium for short-term preservation of veins in cardiovascular operation.

Modulation of monocyte adherence to endothelial cells by endothelin-1 involvement of Src (p60src) and JAK1-like kinases

PURPOSE The purpose of this study was to determine the transmembrane signaling pathway by which endothelin-1 (ET-1) enhances monocyte adherence to human umbilical vein endothelial cells (HUVECs) and to investigate the role of tyrosine kinases in this mechanism. METHODS Adherence of purified human blood monocytes to HUVEC monolayers was assessed with radiolabeled monocytes. Tyrosine kinase activation was examined by immunoprecipitation and Western blotting. RESULTS ET-1 potentiated monocyte adherence to HUVECs in a biphasic manner with peaks at 10(-10) mol/L and 10(-7) mol/L. A potent antagonist to ET B receptors, when used alone, had no effect. However, the antagonist, when combined with ET-1, significantly enhanced monocyte adherence to HUVECs. Incubation of ET-1 (10(-12) mol/L to 10(-7) mol/L) with HUVECs activated tyrosine kinases in a biphasic manner as identified by immunoblotting with PY20 antibody to tyrosine phosphorylated proteins. Phosphorylated proteins with Mr 60, 110, and 130 kDa were observed after ET-1 stimulation of HUVECs. Of interest, ET A or ET B receptor antagonists failed to antagonize the effect of ET-1. Rather, these receptor antagonists significantly augmented ET-1 induced tyrosine phosphorylation in HUVECs. Immunoprecipitation with antibodies to p60SRC and JAK1 kinases followed by immunoblotting with PY20 antibody suggested that ET-1 receptor response coupling in HUVECs involves the activation of p60SRC and JAK1-like kinases. CONCLUSIONS These data suggest an association between activation of p60SRC and JAK1-like kinases and monocyte adherence in response to ET-1. ET-1-induced monocyte adherence is upregulated by ET B receptor antagonist, suggesting a negative feedback on cell adhesion through this receptor.

Technique for assessment of leukocyte adherence to human umbilical vein endothelial cell monolayers.

The interaction between endothelial cells and immune/inflammatory cells plays an important role in the pathogenesis of vascular diseases. Inflammatory cells also activate endothelial cells and release both proliferative and cytotoxic mediators. In order to examine the interaction between leukocytes and endothelial cells and the effect of various drugs, we established the methodology for isolating and culturing the endothelial cells from human umbilical vein. Endothelial cells were harvested by using 0.1% collagenase within 48 hr of collecting the cord. Cells were grown to confluency in 96-well plates in Medium 199 containing 20% fetal calf serum, endothelial cell growth supplement, heparin, and antibiotics. Using this method, we obtained a confluent layer of the cells in all the 96 wells within 48 hr. We then examined the effect of peptides, endothelin-1, substance P, and neurokinin-A on the adherence of human blood neutrophils (purity and viability > 98%) to endothelial monolayers. All the peptides enhanced (p < 0.05) the adherence of neutrophils to endothelial cells in a time-dependent manner. This method of endothelial cell culturing is reliable, reproducible, and effective in evaluating the role of various mediators and drugs on the adherence of various white blood cells to endothelium.

Endothelin-1 levels in ischaemia, reperfusion, and haemorrhagic shock in the canine infrarenal aortic Revascularisation model

Endothelin-1 (ET-1) is a potent vasoconstrictive polypeptide produced from vascular endothelial cells. The effects of ischaemia, reperfusion, and exsanguination on plasma ET-1 levels were studied and compared in the mongrel dog after infrarenal aortic cross clamping. Ischaemia produced a trend toward increased ET-1 serum levels (p < 0.07 with Bonferroni correction) that did not reach significance. Plasma ET-1 levels were significantly increased during reperfusion and even further elevations were found following exsanguination. We found a 2-3 fold increase in ET-1 levels following reperfusion (Initial 3.19 +/- 0.27 pg/ml vs. Reperfusion maximum 6.32 +/- 0.72 pg/ml, Bonferroni p < 0.01). Haemorrhagic shock was associated with a 3-4 fold increase in ET-1 levels (Initial 3.19 +/- 0.27 pg/ml vs. Exsanguination maximum 8.37 +/- 0.97 pg/ml Bonferroni p < 0.001). These data reveal that ET-1 is released during reperfusion and exsanguination and may mediate remote vascular events associated with infrarenal aortic cross clamping and acute blood loss.

G-proteins in rat blood vessels—I. identification☆

1. The purpose of this investigation was to identify various types of conventional, low and high molecular weight G-proteins in purified membranes of rat aorta and mesenteric artery. 2. In both blood vessels, immunoblotting of G-proteins using AS/7 antibody specific for Gi-1/2, EC/2 antibody specific for Gi-3 and RM/1 antibody specific for Gs-type conventional G-proteins demonstrated the presence of M(r) 28-43 kDa, M(r) 41 and 48 kDa, and M(r) 36-46 kDa polypeptides, respectively. Immunoblotting using a common antibody (GA/1) for the Gs and Gi alpha-subunits also revealed the existence of multiple polypeptides (M(r) 24-52 kDa) in both aorta and mesenteric artery, some of which were identified by the specific antibodies. The intensity and number of bands detected by most of the antibodies were greater in mesenteric artery than in aorta. 3. Cholera toxin (CT) catalyzed ADP-ribosylation of two Gs alpha (M(r) 43, 46 kDa) in both types of blood vessels with similar intensities of bands. Pertussis toxin (PT), on the other hand, catalyzed ADP-ribosylation of one Gi alpha (M(r) 41 kDa) polypeptide, and the intensity of this band was greater in aorta than in mesenteric artery. The polypeptides ADP-ribosylated by the toxins corresponded with their identification by antibodies. 4. Nitrocellulose blot overlay with [35S]GTP gamma S identified at least seven low molecular weight G-proteins (M(r) 21-30 kDa) in both aorta and mesenteric artery, with greater intensity of bands in mesenteric artery.(ABSTRACT TRUNCATED AT 250 WORDS)

Posttraumatic innominate artery aneurysm with occlusion of the common carotid artery at its origin by an intimal flap

Blunt trauma involving the innominate and carotid arteries is a rare occurrence that can be lethal or have serious neurologic sequelae. To our knowledge this is the first reported case in the international literature describing the association of posttraumatic innominate, artery aneurysm with total occlusion and thrombosis of the common carotid artery at its origin by an intimal flap. The diagnostic problems created by this unusual injury are discussed. In this case the patency of the distal portion of the common and internal carotid arteries was demonstrated by magnetic resonance angiography (MRA), whereas color duplex and digital arteriographic studies were unsuccessful. This demonstration was crucial to patient management. Since no studies are available comparing color duplex imaging, conventional arteriography, and MRA in the evaluation of blunt carotid trauma, this case study is presented to demonstrate the utility of MRA in emergency situations. In addition, we analyze the possible pathogenesis and discuss the surgical treatment.