Modulatory effect of glucose-6-phosphate dehydrogenase deficiency on benzo(a)pyrene toxicity and transforming activity for in vitro-cultured human skin fibroblasts.

Human skin fibroblasts isolated in vitro from subjects carrying the Mediterranean variant of glucose-6-phosphate dehydrogenase exhibit an 85% decrease of this enzymatic activity. There is a 26% and a 94% decrease of the hexose monophosphate shunt and of the reduced nicotinamide adenine dinucleotide phosphate/nicotinamide adenine dinucleotide phosphate ratio, respectively. Incubation with 0.1 mM methylene blue activates the hexose monophosphate shunt 7 times that of normal fibroblasts and only 2.2 times that of glucose 6-phosphate-deficient cells. This behavior is coupled with an increase of the resistance to cell death induced by benzo(a)pyrene, a carcinogen, the activation of which proceeds through a reduced nicotinamide adenine dinucleotide phosphate-dependent arene oxide formation. In contrast, no difference between the normal and the deficient fibroblasts exists as regards the toxic effect of methylnitrosourea, a carcinogen that does not need metabolic activation. A growth-retarding effect of benzo(a)pyrene was observed in both normal and deficient cells during 9 days in vitro. This effect is lower in the fibroblasts carrying the Mediterranean glucose-6-phosphate dehydrogenase variant. Glucose-6-phosphate dehydrogenase deficiency protects human fibroblasts against the benzo(a)pyrene-induced in vitro transformation. This effect is mimicked by the incubation of normal fibroblasts with dehydroepiandrosterone, a strong inhibitor of glucose-6-phosphate dehydrogenase. The deficiency of this enzymatic activity, either genetically transmitted or induced by dehydroepiandrosterone, is coupled with a reduced rate of benzo(a)pyrene conversion to water-soluble metabolites by human skin fibroblasts.

Inhibition of promotion and persistent nodule growth by S-adenosyl-L-methionine in rat liver carcinogenesis: role of remodeling and apoptosis.

The resistant hepatocyte model (initiation/selection) and the triphasic model (initiation/selection followed by phenobarbital, for a maximum of 16 weeks) were compared for their ability to generate enzyme-altered foci (EAF) and nodules in the liver of Wistar rats initiated by diethylnitrosamine. The effects of S-adenosyl-L-methionine (SAM) on the development of preneoplastic tissue was tested in these experimental models. In the absence of phenobarbital (PB), EAF and early nodules (EN) went through a phase of rapid growth, between 4 and 9 weeks after initiation, to a phase in which progressive decrease in number and size occurred. By the 26th week only a few remodeling EAF and nodules were found. In PB-treated rats a rapid increase in the percentage of liver occupied by EAF and EN, up to the 9th week after initiation, was followed by a period of slow growth (from the 9th to the 20th week) and then, after PB withdrawal (20th week), by a drop in the number and size of EAF and EN. However, at the 26th week actively growing nodules with a low tendency to spontaneous remodeling (persistent nodules) developed. EAF and EN showed a high DNA synthesis 5 weeks after initiation. Thereafter, progressive decline in DNA synthesis, coupled with remodeling and decrease in number of biochemical markers, was seen both in the absence and, even though to a lesser extent, in the presence of PB, indicating that preneoplastic lesions became increasingly insensitive to PB. Relatively few apoptotic bodies could be observed in EAF and EN during PB treatment. After PB withdrawal, decrease in growth potential was coupled with increase in apoptotic bodies. In contrast, in persistent nodules relatively high apoptosis occurred which partially counterbalanced high DNA synthesis. Administration of SAM for a maximum of 16 weeks, starting at the 4th week after initiation, caused a great decrease in number and size of EAF and EN, associated with inhibition of DNA synthesis, high cell death by apoptosis, high remodeling, and loss of biochemical markers, in preneoplastic lesions of both PB-treated and untreated rats. A 1-8-week SAM treatment, started after the development of persistent nodules, caused a great regression of nodular lesions, coupled with a sharp fall in DNA synthesis and increase in apoptosis. It is suggested that inhibition by SAM of the development of preneoplastic tissue is linked to a shift of the equilibrium between cell production and cell death in favor of cell death.(ABSTRACT TRUNCATED AT 400 WORDS)

Local delivery of human tissue kallikrein gene accelerates spontaneous angiogenesis in mouse model of hindlimb ischemia.

BACKGROUND: Human tissue kallikrein (HK) releases kinins from kininogen. We investigated whether adenovirus-mediated HK gene delivery is angiogenic in the context of ischemia. METHODS AND RESULTS: Hindlimb ischemia, caused by femoral artery excision, increased muscular capillary density (P:<0.001) and induced the expression of kinin B(1) receptor gene (P:<0.05). Pharmacological blockade of B(1) receptors blunted ischemia-induced angiogenesis (P:<0.01), whereas kinin B(2) receptor antagonism was ineffective. Intramuscular delivery of adenovirus containing the HK gene (Ad. CMV-cHK) enhanced the increase in capillary density caused by ischemia (969+/-32 versus 541+/-18 capillaries/mm(2) for control, P:<0.001), accelerated blood flow recovery (P:<0.01), and preserved energetic charge of ischemic muscle (P:<0.01). Chronic blockade of kinin B(1) or B(2) receptors prevented HK-induced angiogenesis. CONCLUSIONS: HK gene delivery enhances the native angiogenic response to ischemia. Angiogenesis gene therapy with HK might be applicable to peripheral occlusive vascular disease.

Renovascular hypertension in bradykinin B2-receptor knockout mice.

We evaluated whether kinins exert a protective action against the development of two-kidney, one clip (2K1C) hypertension, a model characterized by an activated renin-angiotensin system in the ischemic kidney and increased expression of the bradykinin (BK) B2 receptor in the contralateral kidney. BK B2-receptor knockout (B2-/-), wild-type (B2+/+), and heterozygous (B2+/-) mice underwent clipping of the left renal artery, with the other kidney remaining untouched. Basal systolic blood pressure (SBP, via tail-cuff plethysmography) was higher in B2-/- mice than in B2+/- or B2+/+ mice (121+/-2 versus 113+/-2 and 109+/-1 mm Hg; P<0.05 for both comparisons). SBP did not change from basal values after sham operation, but it increased in mice that underwent clipping. The increase in SBP was greater in 2K1C B2-/- mice than in B2+/- or B2+/+ mice (28+/-2 versus 14+/-2 and 14+/-2 mm Hg, respectively, at 2 weeks; P<0.05 for both comparisons). Blockade of the BK B2 receptor by Icatibant enhanced the pressure response to clipping in B2+/+ mice (29+/-2 mm Hg at 2 weeks). Intra-arterial mean blood pressure (MBP) was higher in 2K1C than in respective sham-operated mice, with the MBP difference being higher in B2-/- mice (32 and 38 mm Hg, at 2 and 4 weeks, respectively), and higher in B2+/+ mice given Icatibant (30 and 32 mm Hg) than in B2+/+ mice without Icatibant (17 and 18 mm Hg). At 4 weeks, acute injection of an angiotensin type 1 receptor antagonist normalized the MBP of 2K1C hypertensive mice. A tachycardic response was observed 1 week after clipping in B2-/- and B2+/- mice, but this effect was delayed in B2+/+ mice. However, the HR response to clipping in B2+/+ mice was enhanced by Icatibant. Within each strain, heart weight to body weight ratio was greater in 2K1C hypertensive mice than in sham-operated control animals (B2-/-: 5.7+/-0.1 versus 5.2+/-0.1; B2+/+: 5.1+/-0.1 versus 4.5+/-0.1; P<0.01 for both comparisons). The clipped kidney weight to nonclipped kidney weight ratio was consistently reduced in mice with 2K1C hypertension. Our results indicate that kinins acting on the BK B2 receptor exert a protective action against excessive blood pressure elevation during early phases of 2K1C hypertension.

Heparin down-regulates the phorbol ester-induced protein kinase C gene expression in human endothelial cells: enzyme-mediated autoregulation of protein kinase C-alpha and -delta genes.

Overexpression of protein kinase C-alpha and protein kinase C-delta has been shown to modulate a number of biological effects, including the cell growth and differentiation. We hypothesized that heparin, a potent antimitogenic drug, could affect the cell proliferation by inhibiting the expression of specific protein kinase C genes. Heparin, markedly but not completely, inhibited the serum-stimulated protein kinase C-alpha and -delta mRNA expression. Protein kinase C inhibition or down-regulation significantly decreased the serum-induced protein kinase C isoenzyme gene expression. Heparin failed to inhibit the residual effect of serum that was resistant to the above-mentioned treatments. Phorbol 12-myristate 13-acetate elicited an increase of protein kinase C isoenzyme gene expression that was completely prevented by protein kinase C inhibition or down-regulation. Heparin dose-dependently counteracted and ultimately abolished the increase in the protein kinase C isoenzyme gene expression elicited by phorbol 12-myristate 13-acetate. These results suggest that the inhibition of an autoregulatory role wielded by protein kinase C on the protein kinase C-alpha and -delta gene expression might represent a possible mechanism by which glycosaminoglycans modulate the cell growth.

Participation of kinins in the captopril-induced inhibition of intimal hyperplasia caused by interruption of carotid blood flow in the mouse.

1. In the rat balloon injury model, angiotensin-converting enzyme (ACE) inhibitors prevent vascular remodelling by inhibiting angiotensin II generation and kinin breakdown. We investigated if ACE inhibition also prevents the structural vascular responses to disruption of carotid artery blood flow and if kinin potentiation plays a role in such a protection. 2. Morphometric analysis of the structural alterations caused by ligation of the left carotid artery was performed 14 days after surgery in J129Sv wild-type mice (B(2)(+/+)) drinking normal tap water or water containing captopril (120 mg kg(-1) per day). In addition, the effect of captopril on vascular remodelling was tested in B(2)(+/+) given the bradykinin (BK) B(1) receptor antagonist des-Arg(9)-[Leu(8)]-BK (DALBK, 50 nmol kg(-1) per day, intraperitoneally) or the BK B(2) receptor antagonist D-Arg, [Hyp(3),Thi(5)D-Tic(7),Oic(8)]-BK (icatibant, 1 micromol kg(-1) per day, intraperitoneally), and in B(2) receptor gene knockout mice (B(2)(-/-)). 3. Interruption of blood flow resulted in carotid artery intimal hyperplasia and media thickening in untreated B(2)(+/+), these responses being partially suppressed by captopril. The inhibition of intimal thickening exerted by captopril was reduced in B(2)(+/+) given DALBK or icatibant (P<0.05 for both comparisons) as well as in B(2)(-/-) (P<0.05). Neither antagonism of kinin receptors nor disruption of the B(2) receptor gene altered the suppressive effect of captopril on media thickening. The protection of vascular wall structure was independent of the reduction in blood pressure by captopril. 4. These results demonstrate that kinins participate in the inhibitory effect of captopril on intimal hyperplasia via B(1) and B(2) receptor signalling. Our findings may have important implications in treating vascular remodelling evoked by altered shear stress conditions.

Correlation between S-adenosyl-L-methionine content and production of c-myc, c-Ha-ras, and c-Ki-ras mRNA transcripts in the early stages of rat liver carcinogenesis.

gamma-Glutamyltranspeptidase (GGT)-positive and glutathione S-transferase (placental-GST-P) positive foci were induced in male Wistar rats by initiation with diethylnitrosamine (DENA), followed by selection and phenobarbital (PB). GGT- and GST-P-positive foci occupied 20-46% and 27-68% of liver parenchyma, respectively, 5-9 weeks after initiation. A high DNA synthesis was found in GGT-positive foci. Decrease in S-adenosyl-L-methionine (SAM) level and SAM/S-adenosylhomocysteine (SAH) ratio, and overall DNA hypomethylation occurred in the liver during the development of enzyme altered foci (EAF). These parameters underwent very small and transient changes in the liver of uninitiated rats at the 5th week, when EAF occupied 0.7-1.4% of the liver. At the 9th week, high RNA transcripts of c-myc, c-Ha-ras, and c-Ki-ras were found in the liver of initiated rats, but not in that of uninitiated rats. Immunohistochemical evaluation of c-myc gene product showed overexpression in GST-P-positive cells. SAM treatment of initiated rats caused inhibition of EAF growth, recovery of SAM/SAH ratio and DNA methylation, and decrease in protooncogene expression proportional to the dose and length of treatment. Liver SAM/SAH ratio was positively correlated with DNA methylation, and negatively correlated with transcript levels of the three protooncogenes. Thus, decrease in SAM/SAH ratio and DNA hypomethylation are early features of hepatocarcinogenesis promotion in rats fed a diet containing adequate lipotrope amounts, paralleled by overexpression of growth-related genes and rapid growth. Re-establishment of a physiologic SAM level makes it possible to inhibit protooncogene expression and EAF growth and to prevent late liver lesion development.

Role of calcitonin gene-related peptide and kinins in post-ischemic intestinal reperfusion.

The involvement of kinins, calcitonin gene-related peptide (CGRP), and tachykinins during mesenteric post-ischemic reperfusion was studied in anesthetized rats by using antagonists for bradykinin (BK) B1, BK B2, CGRP1, or tachykinin NK1 receptor, or by capsaicin-induced desensitization. B1, B2, or CGRP1 receptor antagonists or desensitization attenuated the transient hypotension and plasma protein and leukocyte infiltration of intestinal wall observed during post-ischemic reperfusion. These effects were abolished by the combination of B2 and CGRP1 blockade as well as by B2 antagonism in capsaicinized rats, while NK1 blockade was ineffective. Our results suggest that kinins and CGRP contribute to systemic vasodilatation and microvascular leakage during mesenteric reperfusion. Pharmacological blockade of these systems could help preventing hypotension and intestinal injury consequent to reperfusion.

Effect of S-adenosyl-L-methionine on the development of preneoplastic foci and the activity of some carbohydrate metabolizing enzymes in the liver, during experimental hepatocarcinogenesis.

gamma-Glutamyltranspeptidase (GGT)-positive foci and glutathione-S-transferase, placental (GST-P)-positive lesions occupied 36% and 54% of liver parenchyma, respectively, in Wistar rats 8 weeks after initiation with diethylnitrosamine, followed by selection. The administration of S-adenosyl-L-methionine (SAM, 384 mumol/kg/day) caused 77% and 42% falls in the percentage of GGT-positive and GST-P-positive lesions, respectively. There also occurred a 46% decrease in labeling index of GGT-positive foci, in SAM-treated rats. These changes were associated with decrease in liver pyruvate kinase (PK), lactate dehydrogenase and glycerol-3-phosphate dehydrogenase. SAM did not affect these enzymatic activities in normal and uninitiated controls, but it caused a consistent increase in initiated rats. Enolase, fructose-biphosphatase and malic enzyme (ME) activities increased in the liver of initiated rats. SAM did not modify significantly these enzymatic activities, either in control or in initiated rats. Glucose-6-phosphate dehydrogenase (G6PDH) was 113% higher in the liver of initiated rats than in uninitiated controls. SAM treatment did not significantly affect this enzymatic activity in uninitiated rats, but caused a great decrease in initiated ones. As expected, there occurred a marked rise in GGT activity in the liver of initiated rats, with respect to controls. SAM caused an increase in GGT activity in normal and uninitiated controls, but it caused a 77% fall in GGT activity in initiated rats, coupled with a 380% rise in remodeling of GGT-positive lesions. Histochemical determination of G6PDH and ME activities showed that in the absence of SAM many preneoplastic lesions expressed higher G6PDH and ME activities than surrounding liver. SAM did not affect ME-positive lesions, while it caused a decrease in the number of G6PDH-positive lesions. Immunohistochemical determination of PK activity, isoenzyme L, showed a decrease in GST-P-positive lesions. Many of these lesions were no longer recognizable as lesions expressing a low PK activity, in SAM-treated rats. However, a relatively small number of GST-P-positive lesions expressing a low PK activity were still present in these rats. These data suggest that glucose channelled into triacylglycerol and pyruvate synthesis decreases in rat liver, during the development of preneoplastic foci, while the production of reducing equivalents and pentose phosphates increases, thus favoring DNA synthesis and detoxification reactions. Decrease in DNA synthesis, in SAM-treated rats, is paralleled by a partial reversion of carbohydrate metabolic features to those present in normal liver.

Comparative effects of L-methionine, S-adenosyl-L-methionine and 5'-methylthioadenosine on the growth of preneoplastic lesions and DNA methylation in rat liver during the early stages of hepatocarcinogenesis.

Male Wistar rats, initiated with diethylnitrosamine (DENA), were subjected to a selection treatment, according to the "resistant hepatocyte" model, followed or not followed by phenobarbital (PB). Rats received, for 3 weeks after selection, 4 i.m. doses (96 mmol/kg) of L-methionine, S-adenosyl-L-methionine (SAM), or 5'-methylthioadenosine (MTA), a SAM catabolite formed during polyamine synthesis or by spontaneous splitting of SAM at physiologic temperature and pH. They were then killed. In some rats, SAM and MTA treatments were started 20 weeks after initiation. The animals were killed 3 weeks later and persistent (neoplastic) nodules (PN) were collected. Some rat groups received 1/2 and 1/4 of the above SAM and MTA doses, or 1/8 of the above MTA dose. SAM and MTA, but not methionine, caused a dose-dependent decrease in number and surface area of gamma-glutamyltranspeptidase (GGT)-positive foci, and in labeling index (LI) of focal cells, coupled with remodeling. SAM and MTA liver contents, SAM/S-adenosylhomocysteine (SAH) ratio and overall methylation of liver DNA were low during the development of GGT-positive foci. SAM, but not methionine, caused a dose-dependent recovery of SAM content and DNA methylation, and a partial reconstitution of liver MTA pool. Exogenous MTA only induced the reconstitution of MTA pool, without affecting SAM level and DNA methylation. Recovery of SAM and MTA pool and DNA methylation was found in the rats subjected to SAM plus MTA, indicating the absence of inhibition of DNA methyltransferases in vivo by MTA. MTA also inhibited liver reparative growth in partially hepatectomized rats, without modifying SAM content and DNA methylation of regenerating liver (RL). A high activity of ornithine decarboxylase (ODC) was found in the liver, during the development of preneoplastic foci, and in PN. This activity was inhibited by SAM and MTA treatments. Although MTA was more effective than SAM, the decrease in ODC activity was coupled with a larger fall in DNA synthesis in SAM-treated than in MTA-treated rats. Thus the antipromotion effect of SAM could not merely depend on its (spontaneous) transformation into MTA. Although MTA production may play a role in the SAM antipromotion effect, other mechanisms could be involved. A role of DNA methylation in the inhibition of growth by SAM is suggested. MTA is a potential chemopreventive agent for liver carcinogenesis.

Thermal stability of lysozyme Langmuir-Schaefer films by FTIR spectroscopy.

Fourier transform infrared spectroscopy has been applied to study the thermal stability of multilayer Langmuir-Schaefer (LS) films of lysozyme deposited on silicon substrates. The study has confirmed previous structural findings that the LS protein films have a high thermal stability that is extended in a lysozyme multilayer up to 200 degrees C. 2D infrared analysis has been used here to identify the correlated molecular species during thermal denaturation. Asynchronous 2D spectra have shown that the two components of water, fully and not fully hydrogen bonded, in the high-wavenumber range (2800-3600 cm-1) are negatively correlated with the amine stretching band at 3300 cm-1. On the grounds of the 2D spectra the FTIR spectra have been deconvoluted using three main components, two for water and one for the amine. This analysis has shown that, at the first drying stage, up to 100 degrees C, only the water that is not fully hydrogen bonded is removed. Moreover, the amine intensity band does not change up to 200 degrees C, the temperature at which the structural stability of the multilayer lysozyme films ceases.

Immunopathology and microvascular lesions in human sympathetic ganglia.

Lumbar sympathetic ganglia surgically removed from adult patients affected by obstructive peripheral arteriopathies are investigated with traditional histology and fluorescent antibody technique. The presence of various pictures of microangiopathy is confirmed, mainly haemodynamic (hyaline-oedematous perivascular thickening) phenomena. Since specific fluorescence is absent from any lesion, the autoimmune pathogenic hypothesis may be discarded.

Nuclear DNA content distribution in five parotid gland tumors. A preliminary report.

Quantitative cytophotometric evaluations of nuclear DNA content were made on epithelial cells obtained from five cases of parotid gland tumors. The measurements were performed on sections stained with Feulgen reaction and, as control of diploid value, adventitious small lymphocytes as well as normal epithelial cells were considered. A fairly good correlation between the type of histogram of DNA values and a progressive degree of atypia was found since particularly high levels of hyperdiploidy were found in more severe tumors.

Effects of lectins on cytoskeleton and morphology or cultured chick embryo fibroblasts.

The microfilaments and microtubules of cultured chick embryo skin fibroblasts were studied in the presence of exogenous lectins by an indirect immunofluorescence technique. Lectin treatment induced modifications in the arrangement of myosin, actin and tubulin, determined depolymerization of the proteins and caused changes in cell shape and size. The results suggest that the interaction between lectins and their specific membrane receptors triggers a series of changes in the cytoskeletal pattern via transmembrana as yet unknown mechanisms and that these are responsible for the alterations in cell shape and size.

Opioid peptide gene expression in the primary hereditary cardiomyopathy of the Syrian hamster. I. Regulation of prodynorphin gene expression by nuclear protein kinase C.

Prodynorphin gene expression was investigated in adult ventricular myocytes isolated from normal (F1B) or cardiomyopathic (BIO 14.6) hamsters. Prodynorphin mRNA levels were higher in cardiomyopathic than in control myocytes and were stimulated by treatment of control cells with the protein kinase C (PKC) activator 1, 2-dioctanoyl-sn-glycerol. Both chelerythrine and calphostin C, two PKC inhibitors, abolished the stimulatory effect of the diglyceride and significantly reduced prodynorphin gene expression in cardiomyopathic myocytes. Nuclear run-off experiments indicated that the prodynorphin gene was regulated at the transcriptional level and that treatment of nuclei isolated from control cells with 1, 2-dioctanoyl-sn-glycerol increased prodynorphin gene transcription, whereas chelerythrine or calphostin C abolished this transcriptional effect. Direct exposure of nuclei isolated from cardiomyopathic myocytes to these inhibitors markedly down-regulated the rate of gene transcription. The expression of PKC-alpha, -delta, and -epsilon, as well as PKC activity, were increased in nuclei of cardiomyopathic myocytes compared with nuclei from control cells. The levels of both intracellular and secreted dynorphin B, a biologically active product of the gene, were higher in cardiomyopathic than in control cells and were stimulated or inhibited by cell treatment with 1,2-dioctanoyl-sn-glycerol or PKC inhibitors, respectively.

Fluorescence cytometry of microtubules and nuclear DNA during cell-cycle and reverse-transformation.

Synchronized CHO-K1 cells and their dibutyryl c-AMP treated counterparts have been characterized by means of static and flow fluorescence cytometry at the level of nuclear DNA and cytoplasmic microtubules. In order to confirm earlier findings on synchronized population, Carnoy fixed and hydrolyzed, several new findings are here reported at the level of single intact cell. The fluorescence intensity of DAPI-stained glutaraldehyde fixed 2C cells correlates well with the average absorbance of the corresponding Feulgen-stained cells, thereby appearing also to be a measure of chromatin condensation during the G1 phase. In the early part of G1, the drastic alteration in anti-beta tubulin immunostaining is shown to parallel microtubule depolymerization induced by calcium or colcemide. The known 1-2 h lengthening of the G1 period after reverse-transformation appears to correlate with a similar delay in the abrupt chromatin decondensation. The above results are discussed in terms of the role of microtubules and nuclear morphometry (and their coupling) in the control of cell cycle progression of transformed vs. fibroblast-like cells.

Rescue of impaired angiogenesis in spontaneously hypertensive rats by intramuscular human tissue kallikrein gene transfer.

Angiogenesis represents a compensatory response targeted to preserve the integrity of tissues subjected to ischemia. The aim of the present study was to examine whether reparative angiogenesis is impaired in spontaneously hypertensive rats (SHR), as a function of progression of hypertension. In addition, the potential of gene therapy with human tissue kallikrein (HK) in revascularization was challenged in SHR and normotensive Wistar-Kyoto rats (WKY) that underwent excision of the left femoral artery. Expression of vascular endothelial growth factor and HK was upregulated in ischemic hindlimb of WKY but not of SHR. Capillary density was increased in ischemic adductor muscle of WKY (from 266+/-20 to 633+/-73 capillaries/mm(2) at 28 days, P<0.001), whereas it remained unchanged in SHR (from 276+/-20 to 354+/-48 capillaries/mm(2), P=NS), thus compromising perfusion recovery as indicated by reduced plantar blood flow ratio (0.61+/-0.08 versus 0.92+/-0.07 in WKY at 28 days, P<0.05). In separate experiments, saline or 5x10(9) pfu adenovirus containing the HK gene (Ad.CMV-cHK) or the beta-galactosidase gene (Ad.CMV-LacZ) was injected intramuscularly at 7 days after the induction of ischemia. Ad.CMV-cHK augmented capillary density and accelerated hemodynamic recovery in both strains, but these effects were more pronounced in SHR (P<0.01). Our results indicate that native angiogenic response to ischemia is impaired in SHR, possibly as a result of defective modulation of endothelial cell mitogens. Supplementation with kallikrein, one of the growth factors found to be deficient in SHR, restores physiological angiogenic response utilitarian for tissue healing. Our discoveries may have important implications in vascular medicine for therapeutic benefit.

Role of the bradykinin B2 receptor in the maturation of blood pressure phenotype: lesson from transgenic and knockout mice.

The binding of bradykinin (BK) to its B2 receptor results in a wide spectrum of biological effects including vasodilation, smooth muscle contraction and relaxation, pain, and inflammation. In order to gain a better insight into the physiological function of this potent vasoactive peptide, murine models have been created by the use of gene insertion or deletion. The results of studies using these strategies are revisited in the present article. In transgenic mice harboring the human BK B2 receptor cDNA (cHBKR), expression of the transgene was identified in the aorta, brain, heart, lung, liver, kidney, uterus and prostate gland by RT-PCR Southern blot analysis. These mice displayed an exaggerated hypotensive response to intra-aortic injection of BK, whereas the blood pressure of knockout mice, homozygous for targeted disruption of the endogenous gene, was insensitive to BK. Two transgenic mouse lines expressing the human BK B2 receptor showed a significant reduction of systolic tail-cuff blood pressure (84 +/- 1 mm Hg, n = 28; 80 +/- 1 mm Hg, n = 24; P < 0.001) compared with the control littermates (97 +/- 1 mm Hg, n = 52). Systolic blood pressure was elevated in BK B2 receptor knockout mice (124 +/- 1 mm Hg, n = 38). In heterozygous mice, systolic blood pressure was similar to that of controls until 5 month-old, then it raised to the elevated levels of knockout mice at 7 months of age. Together these data indicate that kinins acting through the B2 receptor play a role in the development of the blood pressure phenotype.