Aluminum absorption and distribution: effect of parathyroid hormone.

In rats, gastrointestinal aluminum absorption and tissue distribution were altered by parathyroid hormone; the resultant tissue concentrations were similar to those observed in dialysis patients with a fatal encephalopathy. In dialysis patients, serum aluminum and endogenous parathyroid hormone concentrations are significantly correlated. These data suggest that aluminum toxicity in dialysis patients results from oral aluminum ingestion in the presence of hyperparathyroidism.

Electromagnetic guidance for catheter-based transendocardial injection: a platform for intramyocardial angiogenesis therapy. Results in normal and ischemic porcine models.

OBJECTIVES: To test the feasibility of myocardial angiogenic gene expression using a novel catheter-based transendocardial injection system. BACKGROUND: Angiogenesis has been induced by direct injection of growth factors into ischemic myocardium during open-heart surgery. Catheter-based transendocardial injection of angiogenic factors may provide equivalent benefit without need of surgery. METHODS: A new guidance system for intramyocardial therapy utilizes magnetic fields and catheter-tip sensors to locate a position in space and reconstruct three-dimensional left ventricular (LV) electromechanical maps without using fluoroscopy. A retractable 27G needle was coupled with the guidance system for LV transendocardial injection. In 12 pigs, the catheter was used to inject 0.1 ml of methylene-blue (MB) dye and 8 pigs had myocardial injections of adenoviral vector (1 x 10(10) particles per site) containing the LacZ transgene. Ten pigs underwent catheter-based transendocardial injection and six pigs were injected using transepicardial approach with the gene encoding adenovirus vascular endothelial growth factor-121 (Ad.VEGF121; 1 x 10(10) viral particles x 6 sites) and sacrificed at 24 h. Injection sites were identified with ultraviolet light by coinjection of fluorescent beads. RESULTS: Overall, 138 of 152 attempted injection MB tracks (91%) were found after sacrifice. Tissue staining was 7.1+/-2.1 mm in depth and 2.3+/-1.8 mm in width. No animal had pericardial effusion or tamponade. In Ad.LacZ injected animals, gross pathology showed positive staining in injected zones, and histology confirmed positive myocyte staining. Adenovirus vascular endothelial growth factor-121 injected sites showed high levels of VEGF121 production that was of similar magnitude whether injected using the transendocardial (880.4+/-412.2 pg VEGF121/mg protein) or transepicardial (838.3+/-270 pg VEGF121/mg protein) delivery approach (p = 0.62). CONCLUSIONS: Using this magnetic guidance catheter-based navigational system, transgenes can effectively be transfected into designated myocardial sites. Thus, if it is determined that direct intramyocardial injection of angiogenic factors enhances collateral function in patients, this less invasive catheter-based system offers a similar gene delivery efficiency and, thus, may have clear advantages compared with the surgically-based transepicardial injection approach.

Angiotensin II and renal functional reserve in rats with Goldblatt hypertension.

We have previously demonstrated that loss of renal functional reserve (renal response to protein loading) in two-kidney, one clip Goldblatt hypertension is characterized by no change in glomerular filtration rate or single nephron glomerular filtration rate and decreased absolute proximal tubular reabsorption during glycine administration. Captopril restores proximal reabsorption and renal functional reserve in this condition. Because captopril suppresses angiotensin II generation and increases bradykinin, prostaglandins, and potentially nitric oxide, we have investigated the role of angiotensin II blockade in restoring proximal reabsorption and renal functional reserve by comparing captopril with DuP 753, an angiotensin II receptor antagonist, in Goldblatt rats. One month after clipping, two period micropuncture studies (control and glycine) were performed on the unclipped kidney. Normal rats and three groups of clipped rats were studied: an untreated group (HYP), a group treated with captopril (CEI), and a group treated with DuP 753 (DuP) 5 days before micropuncture. Glycine increased glomerular filtration rate, nephron plasma flow, and single nephron glomerular filtration rate in normal rats. Systemic and glomerular hypertension in HYP rats was associated with loss of renal functional reserve and a decrease in absolute proximal reabsorption during glycine. Captopril and DuP 753 normalized systemic and glomerular capillary pressure and prevented the decrease in proximal reabsorption during glycine; however, only CEI rats increased single nephron glomerular filtration rate and glomerular filtration rate after glycine. In conclusion, abnormal responses of both glomerular and tubular function are responsible for the loss of renal functional reserve in Goldblatt rats. Inhibitory angiotensin II activity is responsible for decreasing proximal reabsorption during glycine; however, factors other than angiotensin II limit the glomerular response to glycine.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of lipoprotein(a) plasma concentration on neointimal growth in a monkey model of vascular injury.

Lipoprotein(a) [Lp(a)] has been proposed as a risk factor for both restenosis and coronary heart disease. Recently, we identified Lp(a) in the arterial wall during the initial rapid neointimal growth phase that occurs after balloon injury in cynomolgus monkeys. The purpose of this study was to determine the relationship between circulating Lp(a) levels and the extent of early neointimal formation. Initially, 348 cynomolgus monkeys were screened to identify 15 monkeys that had either high or low circulating Lp(a) levels. In the 15 monkeys, circulating Lp(a) levels were confirmed by two separate measurements over 6 weeks using an immunoturbidimetric assay. Cohorts were identified with plasma Lp(a) levels that differed by four fold. Lp(a) levels expressed as total mass averaged 32 +/- 4 (N = 8) and 136 +/- 12 (N = 7) mg/dl in the low and high groups, respectively. Between the two assays absolute Lp(a) levels differed by less than 6%. Iliac arteries were harvested 14 days after injury induced by expansion of the internal vessel diameter 1.4 times its initial size with an angioplasty balloon. The neointimal area in the high Lp(a) monkeys was 16% greater (0.49 +/- 0.12 mm2, N = 8 versus 0.57 +/- 0.10 mm2, N = 7) than in the low animals; however, this difference was not statistically significant (P = 0.63). Medial areas averaged 1.27 +/- 0.11 and 1.44 +/- 0.20 mm2 (P = 0.48) in these groups, respectively. Tissue Lp(a) quantification, using a mouse monoclonal anti-Lp(a) antibody, indicated that the percent total area staining positive for Lp(a) was 1.7-fold higher in the high versus the low Lp(a) group (2.7 +/- 0.4% versus 1.6 +/- 0.4%, N = 6-8); this difference was not statistical significant (P = 0.28). In summary, a four-fold increase in circulating plasma Lp(a) levels did not result in a statistically significant enhanced neointimal formation at 14 days after balloon injury. This finding suggests that studies of longer duration may be needed to amplify the trend toward increased neointimal growth observed in this study.

Thiophene systems. 9. Thienopyrimidinedione derivatives as potential antihypertensive agents.

A series of thieno[3,4-d]-, thieno[3,2-d]-, and thieno[2,3-d]pyrimidine-2,4-diones with (phenylpiperazinyl)alkyl substitution at N-3 have been synthesized and evaluated for antihypertensive effects in spontaneously hypertensive rats (SHR). These 49 compounds were compared to the vasodilator standards prazosin and the isosteric quinazoline-2,4-dione SGB 1534. Substitution at the 2-, 3-, or 4-position of the phenyl ring was examined, with that at the 2-position more potent than 4-substitution while the isomeric 3-substituted compounds were least potent. Neither alkylation nor acylation at the N-1 position improved the antihypertensive effects as compared to hydrogen. The three thienopyrimidine-2,4-diones (3-5) that contain a [(2-methoxyphenyl)piperazinyl]ethyl moiety at N-3 and hydrogen at N-1 were found to be potent oral antihypertensive agents in the SHR with doses (mg/kg, po) for reducing systolic blood pressure (SBP) by 50 mmHg (ED-50SBP) of 0.21, 0.19, and 1.0, respectively. The compounds 1-5 were further evaluated for alpha blocking potency by measuring the iv doses necessary to antagonize the phenylephrine pressor response by 50% (ED50) in the SHR. The ED50 values (micrograms/kg) are 10.4, 3.3, 1.7, 2.1, and 15.4, respectively. These results clearly show that all three thiophene systems have potent activity as antihypertensive agents and that 3 and 4 are more potent than 1 or 2 as alpha 1-antagonists in vivo.

Butenolide endothelin antagonists with improved aqueous solubility.

Continued development around our ETA-selective endothelin (ET) antagonist 1 (CI-1020) has led to the synthesis of analogues with improved aqueous solubility profiles. Poor solubility characteristics displayed by 1 required a complex buffered formulation in order to conduct iv studies. To overcome the use of specific iv formulations for preclinical studies on additional drug candidates, analogues with improved aqueous solubility were desired. Several analogues were synthesized with substitution patterns that allowed for the formation of either acid or base addition salts. These derivatives had dramatically improved aqueous solubility. In addition, these analogues retained equivalent or improved ETA receptor selectivity and antagonist potency, versus 1, both in vitro and in vivo. Compound 29, which contains as a substituent the sodium salt of a sulfonic acid, has an ETA IC50 = 0.38 nM, ETA selectivity of 4200-fold, and ETA functional activity of KB = 7.8, all of which are similar or superior to those of 1. Compound 29 also has vastly superior aqueous solubility and solubility duration, compared to 1. Furthermore, 29 after iv infusion displays improved activity to 1 in preventing acute hypoxia-induced pulmonary hypertension in rats with an ED50 = 0.3 microg/kg/h.

Structure-activity relationships in a series of orally active gamma-hydroxy butenolide endothelin antagonists.

The design of potent and selective non-peptide antagonists of endothelin-1 (ET-1) and its related isopeptides are important tools defining the role of ET in human diseases. In this report we will describe the detailed structure-activity relationship (SAR) studies that led to the discovery of a potent series of butenolide ETA selective antagonists. Starting from a micromolar screening hit, PD012527, use of Topliss decision tree analysis led to the discovery of the nanomolar ET(A) selective antagonist PD155080. Further structural modifications around the butenolide ring led directly to the subnanomolar ETA selective antagonist PD156707, IC50's = 0.3 (ET(A)) and 780 nM (ET(B)). This series of compounds exhibited functional activity exemplified by PD156707. This derivative inhibited the ETA receptor mediated release of arachidonic acid from rabbit renal artery vascular smooth muscle cells with an IC50 = 1.1 nM and also inhibited the ET-1 induced contraction of rabbit femoral artery rings (ETA mediated) with a pA2 = 7.6. PD156707 also displayed in vivo functional activity inhibiting the hemodynamic responses due to exogenous administration of ET-1 in rats in a dose dependent fashion. Evidence for the pH dependence of the open and closed tautomerization forms of PD156707 was demonstrated by an NMR study. X-ray crystallographic analysis of the closed butenolide form of PD156707 shows the benzylic group located on the same side of the butenolide ring as the gamma-hydroxyl and the remaining two phenyl groups on the butenolide ring essentially orthogonal to the butenolide ring. Pharmacokinetic parameters for PD156707 in dogs are also presented.

Use of fluorescent microspheres to localize in vivo gene transfer injection sites.

The potential for using gene therapy to treat a variety of disease states is growing rapidly. Many vector types and delivery systems have been developed that allow the optimization of protein production levels and kinetics for a given therapeutic gene product. In cases in which a transient, localized delivery of gene product is desired, any determination of the locale of transfected tissue by non-marker genes is problematic. We describe a technique by which the use of fluorescent microspheres can help in identifying potentially transfected tissue. Adenovirus containing the gene for beta-galactosidase (beta-gal) was mixed with fluorescent microspheres and injected into rat skeletal muscle and porcine myocardium. The injection sites could be visualized under ultraviolet light and correlated with beta-gal enzyme expression. This method is simple, inexpensive and generally useful for in vivo gene transfer experiments.

Rat sponge implant model: a new system for evaluating angiogenic gene transfer.

Therapeutic angiogenesis, either by protein injection or gene therapy, holds considerable promise for the treatment of coronary and peripheral artery diseases. Given the large number of angiogenic genes available, a simple, well defined, standard system to compare the relative angiogenic efficacy of such genes would be valuable. We have employed a replication-deficient adenovirus vector (complete E1a-, partial E1b- and partial E3-) to deliver the beta-galactosidase (beta-gal, AdLacZ) reporter gene or the human VEGF121 gene (AdGV VEGF121.10) to a rat sponge implant model of angiogenesis. beta-gal staining results reveal a transfection efficiency as high as 60% 24 h after 2x1010 particle units AdLacZ injection. Our results also indicate that a single injection of 2x1010 particle units of AdGVVEGF121.10 in the sponge results in >10, 000 pg VEGF protein expression per milligram of sponge tissue 24 h later. VEGF121 protein concentrations decreased 10-fold within 3 days and 100-fold within 7 days after injection. Significant VEGF121 protein levels were still detectable 14 days after initial virus injection. The high level of gene transfection efficiency was accompanied by enhanced angiogenesis in the sponge, a tissue devoid of any vessels before implantation. Compared to control (AdNull: adenovirus vector without the VEGF gene), AdGVVEGF121.10 induced a 2- to 3-fold up-regulation of angiogenesis at 7 and 14 days post vector injection as determined by both increased capillary number and increased tissue ingrowth. The angiogenic effects of AdGVVEGF121. 10 were dose-related in this model system. These findings demonstrate a dose-related angiogenic response to adenovirus-mediated gene therapy in this model.

Restenosis: is there a pharmacologic fix in the pipeline?

One of the most frustrating aspects of restenosis is that it is the result of advances in medical care (there was no restenosis before the days of balloon angioplasty), yet it seems to be resistant to all that science has to offer. Still we believe there is reason to be optimistic. We are at last beginning to see some promise from clinical trials, and data being generated confirm some of the hypotheses previously generated from animal experiments. Thus the effects seen with the GP IIb/IIIa antibody 7E3 suggest that thrombosis may be as important in its long-term sequelae as it is for acute reocclusion. The jury is still out on whether antiproliferative approaches will be a therapeutic option, but local delivery paradigms using novel formulations delivered by catheter or impregnated in stents may allow the concept to be tested without the risk of systemic toxicity. Plans are also underway for gene therapy trials, although we may have to wait for better vector technology before taking these into the coronary bed. Perhaps we should move away from the "single pill" approach and accept that, like many infections, malignancies, or even heart failure, a multifaceted approach with combination therapy will provide the first glimmer of that brighter tomorrow.

Hemodynamic effects of epidermal growth factor in conscious rats and monkeys.

The therapeutic application of growth factors to human disease has become closer to reality with the advent of faster means of synthesizing these molecules and novel drug delivery strategies. Epidermal growth factor (EGF) belongs to a large family of molecules with the ability to modulate growth. Purified extracts of EGF have been used clinically to modulate gastrointestinal secretion of hormones and accelerate healing. EGF is also reported to have both vascular smooth muscle contractile and relaxing activity Cardiovascular studies were performed with the bioactive 48-amino acid fragment of human EGF in rodents and primates to determine the effects of EGF on blood pressure and heart rate in conscious animals. Intravenous infusion of EGF induced an initial pressor response in rats followed by a prolonged decrease in blood pressure. In contrast, in monkeys, EGF had dose-related blood pressure-lowering effects only; significant hypotension was observed at doses ranging from 3 to 300 microg/kg i.v. Hypotension was associated with modest tachycardia in both species. To our knowledge, this is the first report of hemodynamic effects of EGF in primates, and it clearly documents that the mitogenic role of growth factors such as EGF is but one aspect of their physiology.

Inhibition of cell growth: effects of the tyrosine kinase inhibitor CGP 53716.

The growth factors, platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) play major roles in enhanced smooth muscle cells growth in rodent blood vessels after vascular injury. Tyrosine kinase inhibition has been shown to be effective in blocking tyrosine phosphorylation at the PDGF and bFGF receptors in cultured fibroblast and vascular smooth muscle cells which in turn inhibits their proliferation. Our study evaluated the PDGF selective tyrosine kinase inhibitor, CGP 53716, on serum, PDGF-BB, bFGF or epidermal growth factor-induced growth responses in cultured rat aortic smooth muscle cells (RASMC) and Balb/3T3 fibroblasts (3T3). CGP 53716 inhibited serum-induced cell growth in RASMC, but not in 3T3 cells. CGP 53716 completely blocked PDGF-BB tyrosine receptor autophosphorylation in RASMC and 3T3 cells, PDGF-BB-induced phosphorylation of mitogen-activated protein kinase at 1 microM in RASMC and inhibited PDGF-BB-induced c-Fos protein expression at 1 microM in RASMC; consistent with inhibition of PDGF-BB-induced DNA synthesis. To examine the selectivity of CGP 53716, PDGF-BB, bFGF or EGF-induced DNA synthesis was measured using thymidine incorporation. CGP 53716 inhibited PDGF-BB-, bFGF- and EGF-induced DNA synthesis in a concentration-dependent manner in each cell line. CGP 53716 showed a 2- to 4-fold selectivity for PDGF-BB-stimulated DNA synthesis over bFGF or EGF in RASMC or 3T3 cells. To rule out that bFGF induced the release of endogenous PDGF, an antibody to PDGF-AB, which binds to all three isoforms of PDGF, was coincubated with bFGF and did not suppress the DNA synthesis induced by bFGF. Based on these results, CGP 53716 is not selective for the PDGF receptor as previously reported. However, EGF-stimulated receptor autophosphorylation of mitogen-activated protein kinase phosphorylation and c-Fos protein expression were not inhibited by CGP 53716 at 1 or 10 microM in RASMC. These findings suggest that CGP 53716 may inhibit multiple growth factor pathways as indicated by inhibition of DNA synthesis. However, these effects must be downstream from the signaling for c-Fos protein expression or use an alternate signaling route. These results further suggest that CGP 53716 may have a therapeutic potential for the treatment of vascular proliferative diseases which are stimulated by not only PDGF but other growth factors such as bFGF and EGF.

Vascular endothelial growth factor upregulates the expression of matrix metalloproteinases in vascular smooth muscle cells: role of flt-1.

Vascular endothelial growth factor (VEGF) is a critical regulator of angiogenesis that stimulates proliferation, migration, and proteolytic activity of endothelial cells. Although the mitogenic activity of VEGF is endothelial cell specific, recent reports indicate VEGF is able to stimulate chemotaxis and tissue factor production in monocytes. VEGF-stimulated activity in monocytes is mediated by the VEGF receptor flt-1. The purpose of the present study was to investigate the effects of VEGF on another major cell type in the vascular wall, namely, the vascular smooth muscle cell (SMC). Using cultured cells, we showed that VEGF has a minimal mitogenic effect on SMCs, which is in accordance with published data. However, VEGF treatment significantly enhanced production of matrix metalloproteinase (MMP)-1, -3, and -9 by human SMCs. The upregulation of MMP-1 and MMP-9 was pronounced, and the stimulation for MMP-3 was less prominent. Stimulation could be demonstrated at both protein and mRNA levels, as reflected by ELISA, zymography, and Northern blot analysis. To explore the signal transduction pathway for the effect of VEGF on SMCs, we studied the expression of 2 high-affinity VEGF receptors, the kinase insert domain-containing receptor (KDR) and flt-1, in human SMCs. Both reverse transcriptase-polymerase chain reaction and immunoblotting revealed the expression of flt-1. Immunoprecipitation followed by immunoblotting illustrated phosphorylation of the flt-1 receptor after VEGF treatment. Similar methodology failed to detect expression of KDR in human SMCs. These data suggest the role of flt-1 in mediating VEGF-stimulated MMP expression of SMCs. The physiological relevance of MMP upregulation was studied by examining VEGF-stimulated SMC migration through 2 synthetic extracellular matrix barriers, Matrigel and Vitrogen. Our results indicate that VEGF treatment accelerated SMC migration through both barriers, and that this response was blocked by MMP inhibition in Matrigel, which supports a permissive role of MMP in SMC migration. These data are the first to show a direct effect of VEGF on SMCs. SMC-derived MMPs may be an additional source of proteases to digest vascular basement membrane, which is a crucial step in the initial stage of angiogenesis. The MMPs may also contribute to SMC migration in angiogenesis and atherogenesis.

Hepatocyte growth factor enhances MMP activity in human endothelial cells.

Scatter factor (SF) or hepatocyte growth factor (HGF) has been identified as an angiogenic factor. Angiogenesis requires not only tube formation but also invasion of pericytes and extracellular matrix (ECM) remodeling to promote new vessel stabilization. In the current study, the effect of SF/HGF on endothelial cell (EC) production of matrix metalloproteinases (MMPs) was explored. We showed that SF/HGF enhanced MT1-MMP synthesis and induced MMP-2 activation in two human EC lines: dermal microvessel EC and coronary arterial EC. Furthermore, SF/HGF accelerated EC invasion into matrix, an activity that could be inhibited by a MMP inhibitor. We also demonstrated that the MAP kinase cascade is critical in signal transduction pathway from SF/HGF stimulation to MT1-MMP up-regulation. The current study indicates that MMP activation is a novel effect of SF/HGF on ECs.

Expression of membrane-type matrix metalloproteinase in rabbit neointimal tissue and its correlation with matrix-metalloproteinase-2 activation.

Smooth muscle cell (SMC) phenotypic alteration, followed by migration and proliferation, is a prominent feature of atherogenesis and vascular neointimal formation. Despite extensive research, mechanism(s) responsible for this alteration remain unclear. Recently, matrix metalloproteinases (MMP), a family of potent proteinases, have been implicated in vascular diseases by way of extracellular matrix degradation. Of particular interest is that expression of a 72-kD MMP (MMP-2) is elevated in neointima, and inhibition of this MMP results in reduced SMC migration and proliferation, suggesting a role for MMP-2 in neointimal development. However, MMP-2 needs activation before digesting protein; the mechanism of this activation in the arterial wall is largely unexplored. A novel membrane-type MMP termed MT-MMP-1 has recently been identified, and its expression in tumor cells is concomitant with MMP-2 activation. Transfection of this MMP cDNA into mammalian cells results in activation of MMP-2. However, the importance of this MMP in various pathological situations is not clear. The present study was designed to explore the relationship between MT-MMP- 1 expression and MMP-2 activation during rabbit neointimal development. Using polymerase chain reaction, we isolated a rabbit cDNA from arterial SMC; sequence analysis indicated that it is a rabbit form ofMT-MMP-1. A segment of this cDNA was subcloned into pGEM-3 and employed to synthesize a DIG-labeled RNA probe. This probe was then used in the Northern blot analysis for MT-MMP-1 mRNA expression both in aortic tissue and in neointimal tissues developed 3, 7, 14 and 21 days after balloon catheter de-endothelialization. The results show low-level expression ofMT-MMP-1 in the normal aortic wall; expression is significantly increased in the neointimal tissues, with peak expression observed in tissues 3 days after injury. Expression of active MMP-2 was also determined using gel zymography. A close temporal expression pattern was observed between MT-MMP-1 and active MMP-2. These data verify the expression of MT-MMP-1 in arterial SMC and suggest its importance in MMP-2 activation after balloon catheter de-endothelialization.

Molecular characterization of rabbit CPP32 and its function in vascular smooth muscle cell apoptosis.

Vascular remodeling in atherogenesis is marked not only by cellular proliferation and migration but is also impacted by apoptotic cell death. Extensive studies have focused on the signal transduction events leading to apoptosis. CPP32, a member of the caspase/interleukin-1 beta-converting enzyme (ICE) protease family, has emerged as a central player in several reports of apoptosis pathways. Vascular smooth muscle cells (SMC) undergo apoptosis after treatment with various stimuli, including nitric oxide (NO) donors, such as sodium nitroprusside (SNP, 0.1-1 mM). The aim of the present study was to evaluate the role of CPP32 in SNP-induced apoptosis of SMC. We isolated a rabbit CPP32 cDNA by using degenerate primers and polymerase chain reaction technique. The predicted protein encoded by this cDNA contains the conserved sequence (QACRG) necessary for covalent linkage to poly(ADP-ribose) polymerase (PARP) as well as the three amino acids responsible for substrate recognition and catalysis reported in other caspase members. Using a segment of this cDNA as a probe, we found no change of CPP32 mRNA in cultured arterial SMC before and after SNP treatment. We also measured the protease activity of CPP32 against a chromophore p-nitroaniline (pNA)-labeled substrate, DEVD-pNA. Our results showed a dose-dependent increase of CPP32 activity in SMC, with a maximal 10-fold increase after SNP treatment. Addition of a competitive CPP32 inhibitor, DEVD-CHO, produced a 50% reduction in maximal stimulation. Immunoblot analysis illustrated that SNP treatment induced proteolytic cleavage of CPP32 into its enzymatically active subunit p17 as well as the degradation of PARP into a 85-kDa fragment. We further demonstrated that incubation of cultured SMC with DEVD-CHO significantly reduced SNP-induced DNA fragmentation. DNA fragmentation analysis was carried out using several methods including a cell death detection enzyme-linked immunosorbent assay kit, in situ end labeling, and DNA electrophoresis in agarose gel. Our data indicate that CPP32 mRNA is constitutively expressed in rabbit SMC and activation of CPP32 protein has a pivotal role in SNP-induced SMC apoptosis.

Clearance of renin in unanesthetized rats: effects of chronic lead exposure.

These experiments studied the influence of chronic lead exposure on the steady-state clearance of exogenous homologous renin in unanesthetized, unrestrained rats. Relative to time controls (TC), rats chronically exposed to 500 ppm lead in drinking water had significantly elevated basal plasma renin concentrations (PRC) (Pb = 12.0 +/- 1.6 ng/ml/hr, TC = 7.6 +/- 0.8). During the infusion of homologous renin sufficient to increase plasma renin approximately 10-fold, the clearance of renin was not different between the two groups (after 60-min infusion, Pb = 16.1 +/- 1.8 ml/kg/min, TC = 15.9 +/- 1.5). Rats exposed to 1000 ppm lead did not have higher PRCs than time controls (Pb = 10.9 +/- 2.2 TC = 9.2 +/- 1.8). The clearances of renin in these two groups were also not significantly different (Pb = 15.3 +/- 2.8 TC = 18.3 +/- 2.5). Renal renin concentrations were significantly elevated in the 500 ppm rats (Pb = 1426 +/- 110 micrograms/kidney, TC = 1065 +/- 118) consistent with an increased basal renin secretion, but were not elevated in the 1000-ppm rats. There were no significant differences in the clearances of sulfobromophthalein (BSP) between any of the groups. The renin infusion significantly reduced BSP clearance, but did not so equally in all groups. It is concluded that in unanesthetized, unrestrained rats the clearance of renin is not altered by chronic lead exposure of either 500 or 1000 ppm and that increased secretion of renin accounts for the elevated basal PRC observed in rats exposed to the lower dose of lead.

Effects of lead on the secretion and disappearance of renin in rabbits.

The disappearance rate of renin from plasma was evaluated in both acutely and chronically lead-exposed rabbits. In addition, the effects of lead (Pb) on in vitro renin secretion were determined with rabbit renal cortical slices. Rabbits acutely exposed to Pb (0.3 to 2.0 mg/kg, iv) demonstrated no increase in plasma renin activity (PRA), but a markedly prolonged disappearance of renin following nephrectomy. Together, these observations suggest that renin secretion must have been inhibited; consistent with this hypothesis was the finding that rabbit renal cortical slices exposed to Pb (10(-5) or 10(-6) M) in vitro secreted significantly less renin than did controls. Thus, the effects of large acute doses of Pb in the rabbit are simultaneous inhibition of both renin secretion and clearance. Chronically Pb-exposed rabbits (500 or 1000 ppm in drinking water) had renin half-lives that were not different from controls (6 to 8 min). PRA was also not significantly different in the three groups. Renal slices from both groups of Pb-exposed rabbits secreted significantly more renin in vitro compared to controls, despite the fact that renal renin concentrations were similar in the three groups. However, the responsiveness to a beta adrenergic stimulus was significantly lower in the slices from rabbits treated with 1000 ppm Pb. Taken together these data suggest that PRA in the chronically Pb-exposed rabbit reflects a tendency for increased basal renin secretion, but a counteracting suppression of renin release secondary to adrenergically mediated stimuli; thus, PRA might be reduced, unchanged, or elevated depending upon experimental conditions. Clearance of renin does not seem to be altered in the chronically Pb-exposed rabbit.

Vascular remodeling in balloon injured rabbit iliac arteries.

To characterize long-term vascular remodeling associated with neointimal formation in vivo, we established a model of balloon injury in normal chow fed rabbits. The iliac artery was injured by denudation using a 2F embolectomy catheter. Injured vessels were removed after perfusion fixation (90 mm Hg) in situ at 2, 4, 6, and 12 weeks post-injury; control vessels were obtained from 2- and 12-week age-matched, uninjured animals. Intimal growth was observed in all animals post-injury. Intimal area averaged 0.13 +/- 0.02 mm2 2 weeks post-injury and continued to increase at 4 and 6 weeks post-injury; +38% and +77% relative to the 2-week time point, respectively. Medical areas were similar among the 2-, 4-, and 6-week injury groups and the 2- and 12-week control groups. From 6 to 12 weeks post-injury, both intimal and medial areas decreased significantly (30% and 34%, respectively); while lumen area increased 53% from 4 to 12 weeks and overall vessel size (area enclosed by the external elastic lamina) remained the same. These data demonstrate that intimal and medial thinning contribute to long term maintenance of lumen area in response to neointimal formation.

Hepatic clearance of renin after angiotensin blockade.

Several investigators have reported that hepatic metabolism of renin can be altered in pathophysiological states (e.g., high-output heart failure, cirrhosis, acute metal toxicity). The hypothesis that circulating angiotensin II may play a role in regulating renin metabolism by the liver was tested in anesthetized dogs. Captopril (SQ 14255) or an angiotensin II-competitive antagonist [( Sar1-Ile8]angiotensin II) was used for blockade of the renin-angiotensin system in two separate groups of dogs. The administration of captopril resulted in a significant fall in the percent extraction of renin by the liver (P less than 0.01) and in the clearance of renin (P less than 0.05). The group receiving the competitive antagonist and time-control animals showed no significant change in renin extraction or renin clearance by the liver. Our data do not support a role for angiotensin II in the regulation of hepatic metabolism of renin, since experiments utilizing the antagonist failed to produce a change. The mechanism by which captopril alters renin metabolism appears to be independent of its blockade of angiotensin II.