Organ-specific changes in vascular reactivity and roles of inducible nitric oxide synthase and endothelin-1 in a rabbit endotoxic shock model.

BACKGROUND: Hemorrhagic shock-induced changes in vascular reactivity appear organ-specific. In the present study, we examined the hypothesis that vascular reactivity induced by septic shock similarly displays organ-specific differences and is regulated by inducible nitric oxide synthase (iNOS) and endothelin-1 (ET-1). METHODS: Endotoxic shock was induced in rabbits by administration of lipopolysaccharide (LPS) (1 mg/kg), and organ specificity of vascular reactivity of superior mesenteric artery (SMA), celiac artery (CA), and left renal artery (LRA) as well as the potential involvement of iNOS and ET-1 examined. RESULTS: Vascular reactivity of SMA, CA, and LRA was increased at the early stages and decreased at the late stages after LPS administration. Superior mesenteric artery showed the greatest decrease in vascular reactivity in response to norepinephrine (NE) (34.9%) and acetylcholine (Ach; 32.3%), followed by LRA (NE, 33.7%; Ach, 30.5%) and CA (NE, 16.2%), whereas the relaxation reactivity of CA in response to Ach was increased to 159%. The mRNA and protein levels of iNOS and ET-1 in SMA, CA, and LRA were not affected at the early stages of endotoxic shock after LPS administration but significantly increased at the late stages. Expression levels were higher in SMA than CA and LRA and negatively correlated with the decrease in vascular reactivity. The iNOS and ET-1 inhibitors, aminoguanidine (20 mg/kg) and PD-142893 (0.02 mg/kg), respectively, induced significant improvements in vascular reactivity and organ perfusion and stabilized the hemodynamic parameters in rabbits subjected to endotoxic shock. CONCLUSION: Changes in vascular reactivity during endotoxic shock are organ-specific. Differential expression patterns of iNOS and ET-1 in different blood vessels contribute to the organ specificity of vascular reactivity. LEVEL OF EVIDENCE: Therapeutic study, level II.

Exogenous glucagon-like peptide-1 acts in sites supplied by the cranial mesenteric artery to reduce meal size and prolong the intermeal interval in rats.

Three experiments were done to better assess the gastrointestinal (GI) site(s) of action of GLP-1 on food intake in rats. First, near-spontaneous nocturnal chow meal size (MS), intermeal intervals (IMI) length and satiety ratios (SR = MS/IMI) were measured after infusion of saline, 0.025 or 0.5 nmol/kg GLP-1 into the celiac artery (CA, supplying the stomach and upper duodenum), cranial mesenteric artery (CMA, supplying small and all of the large intestine except the rectum), femoral artery (FA, control) or portal vein (PV, control). Second, infusion of 0.5 nmol/kg GLP-1 was tested after pretreatment with the GLP-1 receptor (GLP-1R) antagonist exendin-4(3-39) via the same routes. Third, the regional distribution of GLP-1R in the rat GI tract was determined using rtPCR. CA, CMA and FA GLP-1 reduced first MS relative to saline, with the CMA route more effective than the others. Only CMA GLP-1 prolonged the IMI. None of the infusions affected second MS or later eating. CA and CMA GLP-1 increased the SR, with the CMA route more effective than the CA route. CMA exendin-4 (3-39) infusion reduced the effect of CMA GLP-1. Finally GLP-1R expression was found throughout the GI tract. The results suggest that exogenous GLP-1 acts in multiple GI sites to reduce feeding under our conditions and that GLP-1R in the area supplied by the CMA, i.e., the small and part of the large intestine, plays the leading role.

A novel gene delivery method transduces porcine pancreatic duct epithelial cells.

Gene therapy offers the possibility to treat pancreatic disease in cystic fibrosis (CF), caused by mutations in the CF transmembrane conductance regulator (CFTR) gene; however, gene transfer to the pancreas is untested in humans. The pancreatic disease phenotype is very similar between humans and pigs with CF; thus, CF pigs create an excellent opportunity to study gene transfer to the pancreas. There are no studies showing efficient transduction of pig pancreas with gene-transfer vectors. Our objective is to develop a safe and efficient method to transduce wild-type (WT) porcine pancreatic ducts that express CFTR. We catheterized the umbilical artery of WT newborn pigs and delivered an adeno-associated virus serotype 9 vector expressing green-fluorescent protein (AAV9CMV.sceGFP) or vehicle to the celiac artery, the vessel that supplies major branches to the pancreas. This technique resulted in stable and dose-dependent transduction of pancreatic duct epithelial cells that expressed CFTR. Intravenous (IV) injection of AAV9CMV.sceGFP did not transduce the pancreas. Our technique offers an opportunity to deliver the CFTR gene to the pancreas of CF pigs. The celiac artery can be accessed via the umbilical artery in newborns and via the femoral artery at older ages--delivery approaches that can be translated to humans.

Expression and functional role of the RhoA/Rho-kinase pathway in rat coeliac artery.

1. Rho-kinase (ROK) stimulation represents a key step in the maintenance of agonist-induced contraction, an effect counteracted by nitric oxide (NO) released from the endothelium. The aim of the present study was to characterize the involvement of ROK in smooth muscle contraction of the rat coeliac artery using functional and expression studies. 2. Rings of rat coeliac artery were mounted in 5 mL myographs containing warmed and oxygenated Krebs' solution. Rings were connected to isometric transducers and data were recorded in a PowerLab system (ADInstruments, Colorado Springs, CO, USA). After a 60 min equilibration period, preparations were precontracted with phenylephrine (1 micromol/L). Endothelial integrity was assessed by treating the vessels with acetylcholine (1 micromol/L). Expression of ROKalpha, ROKbeta and RhoA was analysed using western blot, whereas Rho guanine nucleotide exchange factors (RhoGEF) were measured at the mRNA level. 3. The addition of Y-27632 (0.01-30 micromol/L) caused sustained relaxation of rings contracted with phenylephrine (PE; 1 micromol/L), with intact or denuded endothelium (pEC50 = 6.38 +/- 0.03 and 5.65 +/- 0.02, respectively). NG-Nitro-L-arginine methyl ester (100 micromol/L) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10 micromol/L), but not indomethacin (10 micromol/L), caused marked rightward shifts of the concentration-response curves to Y-27632. The contractile response to KCl (80 mmol/L) was significantly reduced by Y-27632, with a maximal inhibition of 57 +/- 6%. Nifedipine (0.1-100 nmol/L) fully blocked KCl-evoked contractions, but only marginally affected those in response to PE (27 +/- 2% maximal inhibition). At 1 micromol/L, Y-27632 also significantly enhanced relaxations to sodium nitroprusside (SNP; 0.0001-1 micromol/L). 4. At 1 micromol/L, SNP (but not 1 micromol/L Y-27632) significantly elevated the cGMP content above basal levels. Coincubation with SNP and Y-27632 increased cGMP levels, but the results were not significantly different from those in the presence of SNP alone. 5. Western blot analysis revealed the protein expression of RhoA, ROKalpha and ROKbeta. The PDZ-RhoGEF, p115RhoGEF and leukaemia-associated RhoGEF (LARG) mRNA expression in coeliac artery was visualized by electrophoresis on agarose gels. 6. The results clearly demonstrate a role for the RhoA/ROK signalling pathway in the regulation of rat coeliac artery smooth muscle contraction. The findings of the present study suggest that endogenous nitric oxide-induced relaxation is mediated, in part, by inhibition of RhoA/ROK signalling in this tissue.

[The effects of Xuezhikang on neointimal proliferation and C-myc gene expression after angioplasty in rabbits].

OBJECTIVE: To investigate the effects of Xuezhikang (XZhK) on the neointimal proliferation and C-myc gene expression after angioplasty in rabbits. METHODS: Angioplasty for atherosclerotic stenosis of celiac arteries was performed in 30 male white rabbits after being fed with cholesterol-supplemented diet for 8 weeks, which were then randomized to control group, high-cholesterol group and XZhK group. After 4 weeks, the local vessels were collected for morphological observation. C-myc mRNA level was measured with RT-PCR and C-myc protein with immunohistochemical analysis. RESULTS: Morphological observation showed that the neointimal area in the XZhK group were less than the control group (P < 0.05), and that in control group were less than that in the high-cholesterol group (P < 0.05). The levels of C-myc mRNA measured with RT-PCR and the percentage of C-myc protein positive cell by immunohistochemical analysis were lower in the XZhK group than in the control and high-cholesterol group (P < 0.05), The levels in the latter two groups showed no difference (P > 0.05). CONCLUSION: XZhK can inhibit the neointimal proliferation and the expression of C-myc gene after angioplasty in rabbits.

Early inflammatory reaction of the rabbit coeliac artery wall after combined intraoperative (IORT) and external (ERT) irradiation.

The present immunohistochemical study of radiation-induced damage in major blood vessels is based on a multidisciplinary study (Schultz-Hector et al., Radiother. Oncol., 38: 205-214, 1996) investigating the combined effect of IORT of the coeliac axis and upper abdominal ERT. The paper describes the sequential changes occurring in the coeliac artery after IORT with 30 Gy, i.e. during and after combined IORT and fractionated ERT (total dose 40 Gy). Within 24 h after IORT, the arterial wall was found to be invaded by TNF-alpha positive macrophages, which later on disappeared within 7-14 days. At 2 days post-IORT, the medical smooth muscle cells were strongly positive for TNF-alpha and remained positive throughout the observation period of 63 days. At 80 days, a comparison of different IORT dose groups showed that TNF-alpha expression after 20 and 30 Gy IORT plus 40 Gy ERT had subsided, while it was still strongly evident after 40 Gy IORT. Negative reactions in sham irradiated animals or animals treated with ERT alone indicate that TNF-alpha expression was caused by IORT. After > 30 days post-IORT, there was increased collagen type I deposition in the adventitia. In two animals receiving the full ERT course, intimal proliferations involving mainly smooth muscle cells were observed. Our findings indicate that some features typical of radiation induced arteriosclerosis such as periarterial fibrosis and intimal proliferations can occur as early as < 60 days postirradiation. Macrophage invasion as well as TNF-alpha expression in medial smooth muscle cells are known to be important steps in the development of spontaneous atherosclerotic lesions. Therefore, early TNF-alpha induction in the arterial wall by a high local dose of X-irradiation may be regarded as one initiating factor of chronic radiation-induced arteriosclerosis.

Thromboxane and prostacyclin synthesis in experimental pancreas transplantation. Changes in parenchymal and vascular prostanoids.

The principal causes of failure of a pancreas transplant are rejection and vascular thrombosis. There is an unusually high attrition rate for pancreas transplants, but study models have been difficult to develop. In a rat model that allows study of acute rejection to the exclusion of nonspecific effects of transplant surgery on the pancreas, in vitro synthesis of prostacyclin (PGI2) and thromboxane A2 (TXA2) by transplanted pancreas and the blood vessels transplanted with it was measured using an RIA for their stable hydrolysis products 6-keto-prostaglandin F1 alpha and thromboxane B2 (TXB2). TXB2 synthesis was significantly greater in allotransplanted pancreas than isotransplanted pancreas from the 5th day after transplantation. Rejection was complete in the allografted group 7-9 days after transplantation. 6-Keto-prostaglandin F1 alpha synthesis was similar in the pancreas for both allografts and isografts. Similar changes were seen in aorta, celiac artery, superior mesenteric artery, and portal vein transplanted with the pancreas. In the transplanted aorta, TXB2 was significantly greater in the allograft group from the third posttransplant day. A group of CsA-treated allografts sampled after 9 days had transplanted pancreatic parenchymal and vascular prostanoid synthesis in the isograft range. The changes in PGI2 and TXA2 synthesis that accompany cellular rejection may mediate vascular failure in rejecting pancreas transplants, and changes in PGI2 and TXA2 synthesis in blood vessels transplanted with the pancreas could promote early vascular thrombosis.