Sternal neoangiogenesis following internal mammary artery devascularization: an experimental model.

AIM: The use of bilateral internal thoracic arteries (BIMA) for coronary artery revascularization is associated with better long-term survival and longer freedom from reoperation. Concerns of deep sternal wound infections and mediastinitis have constantly emerged with the utilization of BIMA grafts on a routine basis, especially in diabetic patients. METHODS: We performed a quantitative evaluation of sternal bone healing and angiogenesis after left (LIMA) or bilateral internal mammary artery (BIMA) ligation two and four weeks after sternotomy in normal and diabetic Sprague-Dawley rats. RESULTS: The BIMA group showed a significant increase in neoangiogenesis two weeks after surgery compared to LIMA and control groups (control: 38.3 ± 5.1 vessels/mm², LIMA: 31.4 ± 3.6 vessels/mm², BIMA: 81.6 ± 7.7 vessels/mm²; P=0.047 and P=0.04, respectively). Four weeks after the procedure, bilateral devascularization was associated with lower microvessel formation when compared to LIMA or control groups (control: 50.4 ± 5.2 vessels/mm², LIMA: 64.6 ± 4.9 vessels/mm²; BIMA: 31.5 ± 4.4 vessels/mm²; P=0.006 and P=0.02, respectively). Diabetic animals showed similar results with lower four weeks microvessel formation. However, there were no significant differences when animals with induced diabetes were compared to the normal euglycemic groups for each procedure performed. CONCLUSION: BIMA ligation promotes an early increase in neoangiogenesis. Progressive sternal consolidation is associated with a significant lower level of capillaries and arterioles in the BIMA group four weeks after ligation. Diabetes did not influence the extent of neoangiogenesis between groups with similar procedures. More important clinical determinants could explain the increase incidence of sternal infection in this specific population.

Regulation of VEGF-induced endothelial cell PAF synthesis: role of p42/44 MAPK, p38 MAPK and PI3K pathways.

1. Vascular endothelial growth factor (VEGF) is a potent angiogenic and inflammatory mediator. We have recently shown that this latter effect requires the activation of Flk-1 receptor and subsequent endothelial cell (EC) PAF synthesis. However, the intracellular events that regulate EC PAF synthesis upon Flk-1 stimulation by VEGF remain to be elucidated. 2. Using specific inhibitors and Western blot analysis, we herein report that in bovine aortic endothelial cells (BAEC), VEGF induces the synthesis of PAF through the cascade activation of Flk-1 receptor, phospholipase Cgamma (PLCgamma), protein kinase C (PKC) and p42/44 mitogen-activated protein kinases (MAPK). 3. Moreover, we demonstrate that VEGF-mediated PAF synthesis requires the activation of p38 MAPK, likely by directing the conversion of lyso-PAF to PAF. 4. Interestingly, we observed that VEGF also promoted the activation of the phosphatidyl inositol-3-phosphate kinase (PI3K) pathway, and that its blockade potentiated PAF synthesis following a VEGF treatment. Consequently, it appears that the PI3K pathway acts as a negative regulator of EC PAF synthesis. 5. Taken together, these results allow a better understanding of the intracellular events activated upon EC stimulation by VEGF, and shed a new light on the mechanisms by which VEGF induces PAF synthesis.

Vascular endothelial growth factor production by isolated rat hepatocytes after cold ischemia-warm reoxygenation.

Inflammatory disturbances in the liver microcirculation have been associated with preservation injury of hepatic grafts. Vascular endothelial growth factor (VEGF), a proinflammatory growth factor released by hepatocytes, acts on sinusoidal endothelial cells, but its implication in graft injury is still unclear. We studied VEGF production by rat hepatocytes after cold ischemia and warm reoxygenation and compared the capacity of University of Wisconsin (UW) and sodium-lactobionate-sucrose (SLS) preservation solutions to maintain this hepatocellular function. Isolated hepatocytes were kept for 0, 24, and 48 hours at 4 degrees C in either solution (cold ischemia), then incubated for 1 to 24 hours at 37 degrees C (warm reoxygenation). We assessed cell viability and production of VEGF messenger RNA (mRNA) and protein. Cell viability decreased in a linear time-dependent fashion by 10% after 48 hours of cold preservation and by an additional 40% after 24 hours of warm culture. Very little VEGF mRNA could be detected after up to 48 hours of simple cold preservation in either solution. However, subsequent warm culture led to a robust and rapid increase in VEGF mRNA expression within the first hour, which declined to close to background levels within 8 to 12 hours in culture. This effect was more important in cells preserved in SLS than UW solution. Similarly, cold preservation alone did not trigger VEGF secretion. VEGF secretion was detected after culturing hepatocytes at 37 degrees C and reached a maximal secretion rate within 12 to 15 hours. However, VEGF production by preserved cells was reduced compared with unstored cells. In conclusion, cold ischemia and warm reoxygenation triggers VEGF mRNA expression by hepatocytes, but subsequent VEGF secretion is partially impaired, suggesting posttranslational defects.

Long-term effects of nonselective endothelin A and B receptor antagonism in postinfarction rat: importance of timing.

BACKGROUND: Some controversy exists as to the effects of endothelin (ET) receptor antagonism on long-term post-myocardial infarction (MI) evolution, particularly as it relates to the timing of the intervention after MI (<24 hours versus 10 days). METHODS AND RESULTS: Sham rats and rats surviving an acute MI for >20 hours (n=301) were assigned to treatment with saline or the nonselective ET(A) and ET(B) receptor antagonist LU 420627 (LU) started <24 hours (early) or 10 days (late) after MI and continued for 100 days. Long-term LU treatment led to increased mortality of rats with large MI, regardless of the timing of initiation of therapy. Early initiation of LU reduced survival from 61% to 16% (P<0.001 versus untreated), and later initiation reduced survival to 36% (P=0.012 versus untreated and P<0.001 versus early initiation). Early initiation of LU led to scar thinning, further left ventricular (LV) dilatation, LV dysfunction, and an excessive rise in right ventricular systolic pressure. Later initiation of LU did not modify scar formation but resulted in LV dilatation and dysfunction compared with the untreated group. Cardiac fibrosis tended to increase in the LU-treated MI groups. LU in the sham group reduced cardiac endothelial constitutive nitric oxide synthase but did not modify the changes that occurred with a large MI. CONCLUSIONS: The use of the nonselective ET(A) and ET(B) receptor antagonist LU results in reduced survival, ventricular dilatation, and dysfunction whether started early or late after MI. Early initiation of LU resulted in scar expansion and a particularly unfavorable outcome.

VEGF stimulation of endothelial cell PAF synthesis is mediated by group V 14 kDa secretory phospholipase A2.

1. Vascular endothelial growth factor (VEGF) is a potent inducer of inflammation, and we have shown that this latter effect is mediated through endothelial cell (EC) PAF synthesis. Since the phospholipid remodelling pathway enzymes (CoA-independent transacylase, CoA-IT; phospholipase A2, PLA2; and lyso-PAF acetyltransferase, lyso-PAF-AT) may participate in PAF synthesis, we assessed their contribution to VEGF-induced PAF synthesis in bovine aortic EC (BAEC) and human umbilical vein EC (HUVEC). 2. VEGF enhanced BAEC and HUVEC PAF synthesis by up to 28 and 4 fold above basal levels respectively. 3. A pretreatment with a CoA-IT and lyso-PAF-AT inhibitor (Sanguinarin; 500 nM) blocked VEGF-induced PAF synthesis by 95%, a specific CoA-IT inhibitor (SKF45905; 10 - 50 microM) was without effect, confirming the crucial role of the PLA2 and lyso-PAF-AT. 4. Treatment with secreted PLA2 (sPLA2) inhibitors which have been shown to inhibit both groups IIA and V sPLA2 (SB203347; 10 microM and LY311727; 100 microM) blocked EC PAF synthesis by up to 90%, whereas selective inhibition of group IIA sPLA2 (LY311727; 1 microM) had no significant effect. 5. RT - PCR and Western blot analyses demonstrated the presence of group V sPLA2 whereas group IIA sPLA2 was undetected in EC. 6. Treatment with cytosolic and calcium-independent PLA2 inhibitors (Arachidonyl trifluoromethyl ketone, Bromoenol lactone, Methyl arachydonyl fluorophosphate, up to 50 microM) did not prevent but rather potentiated the VEGF effect on EC PAF synthesis. 7. These results provide evidence that with VEGF activation of EC cells, the group V sPLA2 provides substrate for EC PAF formation.

Coronary artery endothelial dysfunction after ischemia-reperfusion and acute untreated rejection in a canine heterotopic heart transplantation model.

BACKGROUND: Acute rejection is a common problem in heart transplantation and may contribute to the development of cardiac allograft vasculopathy. This study was designed to evaluate the mechanisms of coronary endothelial dysfunction associated with ischemia-reperfusion and acute untreated rejection. METHODS: Two groups of mongrel dogs (n=7 per group) underwent heterotopic cervical heart transplantation without immunosuppression. Allografts were harvested on posttransplant day 1 (group 1) and day 5 (group 2). A third group of unoperated dogs served as control (group 3). After harvesting, epicardial coronary arteries were studied in organ chamber for endothelium-dependent and independent reactivity. RESULTS: Group 1 displayed multifocal ischemic damage without any rejection while hearts from group 2 reached grade IV rejection. Immunohistochemical studies for von Willebrand factor showed expression on coronary endothelial cells in all animals with scattered areas of denudation in transplanted groups. Endothelium-dependent responses to acetylcholine, calcium ionophore A23147, and bradykinin were unaffected in groups 1 and 2. Endothelial relaxations to sodium fluoride (Gi-protein activator) was significantly reduced in group 1 and significantly increased in group 2 compared with control. Responses to serotonin and UK14304 (receptors linked to Gi-protein) were significantly increased in group 2. Responses to thrombin were decreased in both groups. Endothelium-independent responses were unaffected. CONCLUSIONS: In the canine model of heterotopic heart transplantation, the early (24 hr) endothelial dysfunction seen after transplantation is specific to the thrombin receptor and the Gi-protein signaling pathway. Acute untreated rejection did not modify the alteration in endothelial reactivity to thrombin but enhanced the sensibility of the Gi-protein signaling pathways.

Bolus endovascular PDGFR-beta antisense treatment suppressed intimal hyperplasia in a rat carotid injury model.

BACKGROUND: Intimal thickening in accelerated arteriopathies relies on the migration of medial vascular smooth muscle cells (VSMCs) and their proliferation within the neointima. Activation of platelet-derived growth factor receptor-beta (PDGFR-beta) expressed in injured VSMCs is responsible for the migration of medial VSMCs to the intima. In the present study, we wanted to assess whether a single local endovascular delivery of antisense PDGFR-beta in injured rat carotid arteries would be sufficient to prevent intimal hyperplasia and how it might contribute to the vascular healing process. METHODS AND RESULTS: A bolus of antisense PDGFR-beta delivered into injured rat carotid arteries reduced PDGFR-beta protein overexpression by >90% from day 3 to 28 after injury. At day 28 after injury, compared with injured untreated carotids, treatment with antisense PDGFR-beta reduced intimal hyperplasia by 58% and medial VSMC migration by 49% and improved vascular reendothelialization by 100% and vascular reactivity (EC(50)) to acetylcholine by 5-fold. CONCLUSIONS: A single-bolus luminal delivery of antisense PDGFR-beta to injured rat carotids reduced intimal hyperplasia, improved the reendothelialization process, and led to the recovery of endothelium-dependent regulation of vascular tone.

Vascular endothelial growth factor effect on endothelial cell proliferation, migration, and platelet-activating factor synthesis is Flk-1-dependent.

Vascular endothelial growth factor (VEGF) is a potent inducer of endothelial cell (EC) proliferation and migration in vitro as well as inflammation in vivo. We showed recently that VEGF effect on vascular permeability was dependent on the synthesis of platelet-activating factor (PAF) by EC. Consequently, we sought to evaluate by antisense knockdown of gene expression the contribution of VEGF receptors (Flt-1 and Flk-1) on these events. VEGF (10(-11) to 10(-8) M) elicited a dose-dependent increase of bovine aortic EC proliferation, migration, and PAF synthesis by up to 2.05-, 1.31- and 35.9-fold above basal levels, respectively. A treatment with two modified antisense oligomers (1-5 x 10(-7) M) directed against Flk-1 mRNA blocked by 100, 91, and 85% the proliferation, migration, and PAF synthesis mediated by VEGF, respectively. A treatment with two antisense oligomers directed against Flt-1 mRNA failed to modulate these activities. The use of placenta growth factor (up to 10(-8) M), an Flt-1-specific agonist, induced only a slight increase (0.6-fold) of PAF synthesis. These data illustrate the crucial role of Flk-1 in EC stimulation by VEGF. The capacity to inhibit the protein synthesis of Flt-1 and Flk-1 by antisense oligonucleotides provides a new approach to block VEGF pathological effects in vivo.

Identification and characterization of a new growth hormone-releasing peptide receptor in the heart.

Hexarelin, a synthetic hexapeptide of the growth hormone-releasing peptide (GHRP) family with strong growth hormone (GH)-releasing activity, features protecting activity against postischemic ventricular dysfunction in hearts from GH-deficient and senescent rats. To document whether hexarelin action is mediated through specific cardiac receptors, perfusion of Langendorff rat hearts with hexarelin and binding studies were carried out. In the Langendorff rat heart system, hexarelin induced a dose-dependent increase in coronary perfusion pressure. Nifedipine, chelerythrine, and bisindolylmaleimide partially inhibited the vasoconstriction induced by hexarelin, suggesting that this effect was mediated at least in part by L-type Ca(2+) channels and protein kinase C. In contrast, diclofenac and 1-(7-carboxyheptyl)imidazole were without effect, suggesting that prostaglandins and thromboxanes were not involved in the coronary vasoconstriction induced by hexarelin. To characterize the hexarelin binding sites in the rat heart, [(125)I]Tyr-Bpa-Ala-hexarelin was used as photoactivatable radioligand in saturation and competitive binding studies. We specifically labeled a hexarelin receptor with an M(r) of 84 000 in rat cardiac membranes. Saturation binding curves revealed a single class of binding sites with a K(d) of 14.5 nmol/L and a density of 91 fmol/mg of protein. Competition binding studies gave an IC(50) of 2.9 micromol/L for hexarelin; MK-0677 and EP51389, both potent GH secretagogues, did not displace the binding of the photoactivatable derivative from rat cardiac membranes. Interestingly, both compounds were devoid of any vasoconstrictive activity. These results suggest the existence of a new class of hexarelin receptor in the heart, whose role in the regulation of the coronary vascular tone is yet to be determined.

Bradykinin metabolism in the postinfarcted rat heart: role of ACE and neutral endopeptidase 24.11.

The respective role of angiotensin-converting enzyme (ACE) and neutral endopeptidase 24.11 (NEP) in the degradation of bradykinin (BK) has been studied in the infarcted and hypertrophied rat heart. Myocardial infarction (MI) was induced in rats by left descendant coronary artery ligature. Animals were killed, and hearts were sampled 1, 4, and 35 days post-MI. BK metabolism was assessed by incubating synthetic BK with heart membranes from sham hearts and infarcted (scar) and noninfarcted regions of infarcted hearts. The half-life (t1/2) of BK showed significant differences among the three types of tissue at 4 days [sham heart (114 +/- 7 s) > noninfarcted region (85 +/- 4 s) > infarcted region (28 +/- 2 s)] and 35 days post-MI [sham heart (143 +/- 6 s) = noninfarcted region (137 +/- 9 s) > infarcted region (55 +/- 4 s)]. No difference was observed at 1 day post-MI. The participation of ACE and NEP in the metabolism of BK was defined by preincubation of the membrane preparations with enalaprilat, an ACE inhibitor, and omapatrilat, a vasopeptidase inhibitor that acts by combined inhibition of NEP and ACE. Enalaprilat significantly prevented the rapid degradation of BK in every tissue type and at every sampling time. Moreover, omapatrilat significantly increased the t1/2 of BK compared with enalaprilat in every tissue type and at every sampling time. These results demonstrate that experimental MI followed by left ventricular dysfunction significantly modifies the metabolism of exogenous BK by heart membranes. ACE and NEP participate in the degradation of BK since both enalaprilat and omapatrilat have potentiating effects on the t1/2 of BK.

Rat arterial wall retains myointimal hyperplastic potential long after arterial injury.

BACKGROUND: Many novel molecular and pharmacological modalities have been proposed for the treatment of accelerated vascular diseases. Yet the fundamental question remains of whether the vessel wall can be treated once only or whether single-dose therapy simply delays the inevitable processes that lead to intimal hyperplasia. Since platelet adhesion and aggregation are critical events in vascular healing, we sought to determine whether the injured blood vessel would retain its myointimal potential after reversal of even prolonged periods of thrombocytopenia. METHODS AND RESULTS: A novel nonimmune method sustained thrombocytopenia and suppressed postinjury neointimal hyperplasia by 88%. Infusion of fresh platelets, even 14 days after initial denuding injury, restored the full neointimal hyperplastic potential. Platelet depletion presumably removed factors chemotactic for vascular smooth muscle cells but had no effect on the overexpression of the platelet-derived growth factor receptor-beta (PDGFR-beta) subunit after vascular injury. In native vessels, 26.5+/-2.5% of medial smooth muscle cells expressed PDGFR-beta. In all animals, medial PDGFR-beta expression doubled 2 weeks after endothelial denudation and was evident in up to 74.5+/-2.5% of the cells forming the neointima. CONCLUSIONS: Thus, though the hyperplastic potential of the injured blood vessel can be delayed with removal of growth stimuli, it is not lost forever, and if the media is not made quiescent, neointimal hyperplasia is simply delayed rather than prevented. These results may have a profound effect on our understanding and treatment of accelerated proliferative vascular diseases.

DNA antisense strategies in the study of receptors for vasoactive peptides, and of growth and wound-healing factors.

Antisense oligodeoxynucleotide technology has contributed greatly to the overall understanding of both mRNA stability as well as translational processes leading to protein synthesis. Arrest of translational processes by DNA antisense strands usually reduces maximal effects of agonists without affecting their apparent affinities in treated isolated vascular or nonvascular preparations. In the present study, examples are given of DNA antisense oligonucleotide-induced repression of receptors for endothelins, kinins as well as of the platelet-derived growth factor. Furthermore, the efficiency of this technology illustrates the roles of protooncogenes (c-myc and c-myb) in wound-healing mechanisms. The overall mechanism of action of these oligomers is described and the relevance of size, chemical alterations and mode of delivery are illustrated. Release of oligophosphorothioates from polymer matrices and gels can produce a prolonged effect in vivo. Antisense oligonucleotides remain essential in experimental models for which receptor antagonists or selective inhibitors of intracellular components are currently unavailable.

VEGF effect on vascular permeability is mediated by synthesis of platelet-activating factor.

Vascular endothelial growth factor (VEGF), a protein synthesized and secreted by a variety of normal and neoplastic cells, serves as an endothelial cell-specific mitogen and a potent angiogenic factor. Intradermal injection of VEGF increases vascular permeability, a critical event in inflammation and angiogenesis. We sought to identify which tissues are responsive to the inflammatory effect of VEGF, to investigate the mechanisms by which VEGF increases vascular permeability, and to establish whether mediators of inflammation such as platelet-activating factor (PAF) play a role in this regard. Intravenous injection of VEGF (0.001-0.1 nmol/kg) into rats induced dose-dependent protein extravasation in vascular beds of certain tissues up to 177% over control levels. In some tissues, the elevation in vascular permeability in vivo was abolished completely by selective PAF-receptor antagonists. Moreover, VEGF (10(-11) to 10(-9) M) was found to increase PAF synthesis in cultured bovine aortic endothelial cells up to 20-fold. Our results suggest that VEGF increases vascular permeability by inducing PAF synthesis.

Antisense oligonucleotide inhibition of PDGFR-beta receptor subunit expression directs suppression of intimal thickening.

BACKGROUND: The elucidation of molecular mechanisms of vascular cell biology has markedly influenced our thinking on the pathophysiology of vascular disease. Antisense oligonucleotide gene therapy has helped identify proteins critical to cell-cycle progression and proliferation and possible therapeutic strategies to combat human disease. This approach, however, has not yet been used to examine the contribution of chemotactic proteins and/or their receptors. Platelet-derived growth factor-BB (PDGF-BB) released from activated platelets adherent to subendothelial connective tissue is a principal smooth muscle cell chemotactic factor. METHODS AND RESULTS: A series of experiments was performed to assess the capacity of antisense oligonucleotides to reduce PDGF-beta receptor subunit (PDGFR-beta) expression and the contribution of PDGFR-beta in neointimal formation. Sustained, direct, and local perivascular administration of two different antisense oligonucleotide sequences complementary to PDGFR-beta mRNA almost completely abolished the expression of PDGFR-beta protein in the intima and media of injured carotid arteries and decreased neointimal formation by 80% and 60%, respectively. Furthermore, neointimal formation correlated precisely with PDGFR-beta expression in an exponential fashion. CONCLUSIONS: Thus, myointimal proliferation depends on both PDGFR-beta overexpression and its activation by PDGF-BB. Removal of either of these two elements can suppress neointimal formation.

c-myc in vasculoproliferative disease.

Antisense oligonucleotides to genes central to cellular proliferation have suppressed smooth muscle cell growth in vitro and in vivo. We now report that although the response of cultured smooth muscle cells to antisense oligonucleotides to c-myc and c-myb is identical, the response of the injured arterial wall to these oligomers depends on the kinetics of gene expression and oligonucleotide delivery. Two different antisense oligonucleotides to each oncogene were administered to the perivascular aspect of injured rat carotid arteries via polymer-based delivery systems. The acute release of antisense oligonucleotides from the Pluronic gels reduced in vitro cell growth 54.8% with c-myc and 56.9% with c-myb. The more sustained release from ethylene vinyl acetate copolymer (EVAc) matrices was slightly less efficient, inhibiting proliferation 47.3% and 43.3%, respectively. However, although both EVAc and Pluronic release of c-myb antisense oligonucleotide sequences inhibited intimal hyperplasia 2 weeks after injury, only the more prolonged EVAc matrix release of antisense oligonucleotide to c-myc was effective. The failure of the short course of c-myc oligomer release from Pluronic gels stemmed from early successful suppression with late loss of regulation and not from inactivation of the antisense oligonucleotide within the polymeric gel. Within 24 hours of injury, Pluronic-based release of c-myc antisense oligomers reduced mRNA levels in the tunica media 2.5-fold and immunocytochemical identification of c-myc expression by 98.8%. As a result, the number of proliferating cells was decreased 6.5-fold 3 days after injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Regional changes in the extravasation of albumin in the canine kidney: comparison of bradykinin and water diuresis.

This report describes the adaptation of the albumin bound Evans blue dye (EB) extraction technique and its use in identifying regional changes in albumin extravasation rates. We present data to justify our technical approach and highlight the use of this method by describing differences resulting from two different models of induced diuresis and natriuresis. Results observed under control conditions (Group 1) are compared to those obtained following the infusion of bradykinin (BK) into the left kidney (Group 2) or hypotonic saline-induced water diuresis (Group 3). EB and water content of tissue samples of cortex (CTX), outer medulla (OM), inner medulla (IM), and papilla (PAP) regions are reported. Under control conditions a significant heterogeneous distribution of EB and water content (wet/dry tissue weight) between zones was observed. Left kidney EB values for the CTX, OM, IM, and PAP in Group 1 were 125 +/- 11, 398 +/- 56, 763 +/- 51, and 741 +/- 52 micrograms EB/g dry tissue and respective wet/dry tissue ratios were 4.48 +/- 0.05, 5.10 +/- 0.19, 7.13 +/- 0.37, and 6.35 +/- 0.32. In Group 2, BK caused a selective increase in cortex EB content to 201 +/- 7 (P < 0.01) micrograms EB/g dry tissue, without altering water content values. Results of EB extraction in Group 3 revealed no change in the CTX but significant increases in the OM, IM, and PAP regions: 576 +/- 40 (P < 0.01), 910 +/- 60 (P < 0.01), and 850 +/- 69 (P < 0.05) micrograms EB/g dry tissue, respectively. Likewise, tissue water content values were unchanged in the CTX but significantly greater in the OM, IM, and PAP: 6.02 +/- 0.22, 8.90 +/- 0.25, and 8.40 +/- 0.17, respectively (P < 0.01, all three values). This technique clearly shows the regional heterogeneity of the renal microvascular network and allows the localization of intrarenal changes in albumin extravasation. This method provides evidence that BK increases albumin extravasation in the cortex only and that changes in the renal medulla are obtained in hypotonic saline-induced water diuresis.

Acute and chronic diuretic treatment selectively affects vascular permeability in the unanesthetized normal rat.

Diuretics are widely used antihypertensive agents, and although their renal actions have been well characterized, the extent of their vascular effects remains to be defined. Because hypertension is associated with numerous vascular complications whose incidences are not always lowered once blood pressure is regulated, this study was undertaken to evaluate the effects of five selected diuretics on capillary permeability to see if they could contribute in some way to these vascular abnormalities. Extravasation of Evans blue dye (EB: 20 mg/kg) injected in the caudal vein of male Wistar rats was used to assess capillary permeability to albumin. Indapamide (0.04 mg/kg), cicletanine (2.0 mg/kg), amiloride (0.3 mg/kg), hydrochlorothiazide (0.5 mg/kg) and furosemide (0.5 mg/kg) were administered by acute i.v. injection or by 10-day "chronic" gavage. EB extravasation was increased in the upper bronchi, lung parenchyma and kidney after acute administration of indapamide (54, 41 and 31%, respectively) and hydrochlorothiazide (45, 41 and 19%, respectively), and increased in all tissues but the duodenum (upper bronchi, lung parenchyma, heart, liver, kidney and muscle; 57-118%) after furosemide. In contrast, capillary permeability was reduced after acute cicletanine in the heart (31%), duodenum (49%) and muscle (58%) and after amiloride in the heart (25%) and muscle (63%). Pretreatment with indomethacin abolished most changes in EB extravasation induced by acute injection of the diuretics. After 10-day gavage, however, changes in capillary permeability were null after amiloride or hydrochlorothiazide treatment, attenuated after cicletanine or furosemide or even reversed after indapamide. Arterial pressure was not affected by diuretic treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of PAF on rat lung vascular permeability: role of platelets and polymorphonuclear leucocytes.

1. The objectives of the present experiments were to assess the contribution of polymorphonuclear leucocytes (PMNLs), platelets and their products such as thromboxane A2 (TxA2), histamine and 5-hydroxytryptamine to platelet activating factor (PAF)-mediated protein extravasation in rat lungs. 2. Intravenous injection of PAF (1.0 and 5.0 micrograms kg-1) increased dose-dependently (up to 7.5 fold) the vascular permeability of the trachea, upper and lower bronchi to Evans blue dye (EB), a marker of albumin extravasation. The permeability of the pulmonary parenchyma was not affected significantly by PAF. 3. Thrombocytopenia induced by administration of the IgG fraction of goat anti-rat platelet serum (APS; 15 mg 100 g-1, i.p., 16-18 h) reduced by 55, 58 and 40% the effects of the lower dose of PAF (1.0 microgram kg-1) and by 31, 23 and 15% the effects of the higher dose of PAF (5.0 micrograms kg-1) on the permeability of the trachea, upper and lower bronchi respectively to albumin. 4. PMNL depletion induced by administration of rabbit anti-rat polymorphonuclear serum (ANS; 2 mg kg-1, i.v., 24 h) did not reduce significantly the effects of the lower dose of PAF (1.0 microgram kg-1) on the airways, however the effects of the higher dose of PAF (5.0 micrograms kg-1) on the permeability of the trachea, upper and lower bronchi to albumin were reduced by 43, 25 and 23% respectively. 5. The injection of both the anti-platelet and the anti-PMNL sera reduced by 61, 62 and 96% the effects of the lower dose of PAF (1.0 microg kg-1) and by 44, 39 and 47% the effects of the higher dose of PAF (5.0 microg kg-1) on the permeability of the trachea, upper and lower bronchi respectively.6. The combined injection of the TxA2-mimetic (U-44069; 5.0 microg kg-1) and PAF (1.0 and 5.0 microg kg-1)in thrombocytopenic rats overcame the vascular permeability decrease induced by APS treatment.7. Pretreatment of the animals with a combination of antagonists to histamine (mepyramine;3.0 mg kg-1) and 5-hydroxytryptamine (methysergide; 2.5 mg kg-1) did not cause a significant inhibition of the effect of PAF (1.0 and 5.0 microg kg-1) on EB extravasation in the airways.8. These data show that the effect of intravenous PAF on rat vascular permeability is partly modulated by polymorphonuclear leucocyte and platelet activation. Our results suggest that following its release,TxA2 could increase postcapillary hydrostatic pressure by inducing a venoconstriction and potentiate the extravasation elicited by PAF. These results do not suggest a major role for histamine and/or 5-hydroxytryptamine on PAF-induced albumin extravasation.

Enhancement by endothelin-1 of microvascular permeability via the activation of ETA receptors.

1. The objective of the present experiments was to assess the involvement of endothelin-A (ETA) receptors in mediating the effects of endothelin-1 on microvascular permeability in conscious rats. 2. Bolus injection of endothelin-1 (0.1 and 1 nmol kg-1, i.v.) resulted in a dose-dependent prolonged pressor effect preceded by a transient depressor response. These changes were accompanied by a dose-dependent loss of plasma volume. Endothelin-1 (1 nmol kg-1) enhanced the vascular permeability of the upper and lower bronchi, kidney, stomach, duodenum and spleen (up to 270%) as measured by the extravasation of Evans blue dye. 3. Pretreatment of the animals with the selective ETA receptor antagonist, BQ-123 (1 mg kg-1, i.v.) significantly blunted the pressor response to endothelin-1 without affecting the depressor response. BQ-123 inhibited by 87% the endothelin-1 (1 nmol kg-1)-induced plasma volume loss. BQ-123 markedly attenuated protein extravasation elicited by endothelin-1 in the upper and lower bronchi and kidney, whereas it completely inhibited the permeability effect of endothelin-1 in the stomach and duodenum. BQ-123 by itself had no significant effect on the parameters studied. 4. The endothelin-1 analogue, [Trp(For)21]-endothelin-1, in which Trp21 is formylated, was as potent a pressor agent as endothelin-1, but had no depressor action. Bolus injection of [Trp(For)21]-endothelin-1 (0.1 and 1 nmol kg-1, i.v.) evoked similar plasma volume losses to those observed following administration of equimolar doses of endothelin-1. Furthermore, 1 nmol kg-1 [Trp(For)21]-endothelin-l evoked increases in protein extravasation similar to endothelin-l, 1 nmol kg-1.5. The present findings suggest that endothelin- 1 enhances microvascular permeability, in part, via the activation of ETA receptors.

Plasma extravasation induced by neurokinins in conscious rats: receptor characterization with agonists and antagonists.

The purpose of the present experiments was to study the effects of various neurokinin related peptides, such as substance P, [beta Ala8]NKA(4-10), and [MePhe7]NKB, which are selective for NK-1, NK-2, and NK-3 functional sites, respectively, to induce plasma extravasation in rats and the effectiveness of RP 67580 and CP-96,345 (two nonpeptide NK-1 receptor selective antagonists) and SR 48968 (a nonpeptide NK-2 receptor selective antagonist) to prevent such an effect. Bolus intravenous injection of substance P (1.0 nmol/kg) into conscious rats induced extravasation of Evans blue dye (EB), a selective marker of albumin vascular permeability, in the duodenum, the stomach, the pancreas, and the urinary bladder by 50, 40, 58, and 312%, respectively; a slight increment occurred also in the ileum and the kidney but was not significant. [beta Ala8]NKA(4-10) (1.0 nmol/kg) increased EB extravasation in the stomach and the urinary bladder by 52 and 99%, respectively, while [MePhe7]NKB (1.0 nmol/kg) did the same in the stomach, the ileum, and the urinary bladder by 58, 50, and 79%. Pretreatment with RP 67580 (250 nmol/kg) blocked the albumin extravasation mediated by substance P in the duodenum, the pancreas, and the urinary bladder by 100, 100, and 78%, respectively. CP-96,345 (250 nmol/kg) also inhibited EB extravasation mediated by substance P in the duodenum and the pancreas by 100 and 100%, respectively, but was ineffective in the urinary bladder. Neither RP 67580 nor CP-96,345 prevented the substance P mediated extravasation in the stomach. RP 67580 and CP-96,345 did not antagonize the effects of NK-2 and NK-3 selective agonists.(ABSTRACT TRUNCATED AT 250 WORDS)