Search

Inappropriate modulation of the renin angiotensin system (RAS) can lead to derangements of blood pressure homeostasis in humans. Cyp1a1-mRen2.F transgenic rats were used to define the renal mechanisms underlying the development of angiotensin II-dependent hypertension. These transgenic rats were previously generated by introducing the mouse Ren2 gene into the rat genome under the control of a Cyp1a1 inducible promoter. The aim of the current investigation was to establish the contribution of renal function to the development of hypertension in the Cyp1a1- mRen2.F transgenic rat. Expression of the mRen2 transgene was induced by daily gavage of indole 3 carbinol (I3C) at the dose of 100mg/kg. Blood pressure was measured in conscious rats after 1, 3 or 7 days of treatment. The control group received the vegetable oil carrier for 7 days. In addition blood pressure, renal haemodynamics and excretory function were measured under thiobutabarbital anaesthesia. Transgene induction caused a progressive increase in blood pressure in a time dependent manner. Neither glomerular filtration rate nor renal blood flow was affected. This indicates proper function of renal autoregulation during the experimental time course. Tubular sodium reabsorption was significantly increased after the first day of transgene induction and this effect was sustained for the duration of treatment. A pharmacological approach was used to localize the increased reabsorption to a specific region of the nephron and was found to reflect increased activity of the thiazide-sensitive cotransporter (NCC). Chronic administration of thiazide significantly blunted the hypertensive response to transgene induction. Similarly AT1 receptor blockade attenuated the hypertensive phenotype and prevented the transgene-induced stimulation of NCC activity. In contrast, mineralocorticoid receptor blockade did not prevent the development of either hypertension or increased NCC activity. The current study suggests that the development of angiotensin II-dependent hypertension is mediated by increased tubular sodium reabsorption. Increased activity of NCC is a key hypertensive mechanism in this model and results directly from the actions of angiotensin II at the AT1 receptor; indirect aldosterone pathways do not play a major role.

Abstract: Background The discovery of angiotensin-converting enzyme 2 (ACE2) introduced an alternative protective arm of the renin-angiotensin system (RAS), the ACE2/Angiotensin 1-7 (Ang 1-7)/Mas receptor axis, to counterbalance the more renowned pathogenic angiotensin-converting enzyme (ACE)/Angiotensin II (Ang II)/AT1 receptor axis that predominates in disease states due to RAS hyperactivation. As a crucial endogenous regulator of the RAS, ACE2 possesses a catalytically active ectodomain exposed to the circulation that hydrolyzes various vasoactive peptides including, Ang II and angiotensin I (Ang I), generating Ang 1-7 and angiotensin 1-9 (Ang 1-9), respectively. Notably, ACE2 has garnered international attention as the cellular receptor for severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2, becoming one of the most researched and well-known human proteins. A soluble form of ACE2 is shed from the membrane through proteolytic cleavage by a disintegrin and metalloprotease 17 (ADAM17), resulting in diminished ACE2-mediated tissue protection under pathological conditions. We propose that the dysregulation of plasma angiotensin peptides and ACE2 is a molecular signature of cardiovascular and inflammatory diseases and can further serve as prognostic markers for adverse clinical outcomes in heart failure (Chapter 2) and coronavirus disease-2019 (Chapter 3). Methods and Results In Chapter 2, we prospectively enrolled 110 patients with heart failure from outpatient clinics and the emergency department to perform comprehensive profiling of circulating and equilibrium levels of plasma angiotensin peptides using liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques. We found that an elevation in the ratio between Ang 1-7 to Ang II, effector peptides of the protective and harmful axis of the RAS, respectively, served as an independent and incremental predictor of beneficial outcomes, better survival rate, and reduced hospitalization duration. Although multiple peptidases participate in the proteolytic processing of Ang 1-7 and Ang II, ACE2 is a direct regulator of the Ang 1-7/Ang II ratio, and its dysregulation contributes to RAS imbalance resulting in adverse clinical outcomes. In Chapter 3, we prospectively enrolled 242 consecutive patients admitted to hospital wards designated for COVID-19 or intensive care units to measure plasma soluble ACE2 and other established disease markers using enzyme-linked immunosorbent assay and angiotensin peptides through LC-MS/MS at admission and repeated sampling on day seven. We found that an upward trajectory in soluble ACE2 was independently associated with an increased risk of mortality and incidence of acute myocardial injury and circulatory shock. In contrast, patients who survived the COVID-19 hospitalization were characterized by a substantial reduction in soluble ACE2. Additionally, we found a prominent elevation in plasma Ang I and Ang 1-7 levels in patients with COVID-19 accompanied by downregulated plasma ACE activity at hospital admission, likely due to the extensive pulmonary vascular endothelial injuries during SARS-CoV-2 infections. Conclusions Heart failure and COVID-19 shares mutual disease pathophysiology in forms of dysregulation in ACE2 and the RAS. Importantly, plasma angiotensin peptide and soluble ACE2 levels can be effectively monitored through disease progression to facilitate a personalized biomarker-guided approach for risk stratification and the implementation of medical therapies against these conditions. Moreover, our data suggest a potential role of ADAM17 inhibition and the development of novel strategies to enhance the beneficial ACE2/Ang 1-7/Mas axis to improve patient outcomes in cardiovascular and inflammatory diseases.

Increased renin-angiotensin-aldosterone system (RAAS) metabolite concentrations instigate pro-fibrotic and pro-inflammatory changes within the kidney, potentially leading to worsening chronic kidney disease (CKD). Vitamin D metabolites have demonstrated the ability to serve as a negative endogenous regulator of the RAAS in humans. CKD, however, leads to decreased serum concentrations of vitamin D metabolites. We hypothesize that supplementation with calcifediol (25-hydroxyvitamin D) will decrease circulating RAAS mediators in dogs with CKD.Six dogs with IRIS Stage 2 and 3 CKD were retrospectively evaluated from a prior study involving the supplementation of calcifediol (25-hydroxyvitamin D). Dogs were supplemented with calcifediol for 3 months, after which calcifediol supplementation was discontinued. Dogs were evaluated at baseline (day 0), month 3, and month 5 (2 months after discontinuation of calcifediol). Vitamin D metabolites (25-hydroxyvitamin D, 1,25 hydroxyvitamin D, 24,25 hydroxyvitamin D) and RAAS mediators (angiotensin I/II/III/IV/1-5/1-7, and aldosterone) were evaluated at all time-points. Calculated surrogate values for plasma renin activity (PRA) and angiotensin-converting enzyme (ACE) activity were also evaluated. RAAS mediators were determined using a novel liquid-chromatography-tandem mass spectroscopy assay (RAS FingerprintTM). There was no significant difference in the concentration of any RAAS parameters between any time-point. The surrogate for ACE activity was significantly lower at month 3 compared to baseline (p = 0.01). No other RAAS mediators were affected by medications at any time-point. In conclusion, short-term calcifediol supplementation in this small subset of CKD significantly impacted ACE activity.

Abdominal aortic aneurysms (AAAs), a permanent dilation in the abdominal region of the aorta, is a highly sexually dimorphic disease. AAAs prevalence is ranging from 4-10 fold higher in males than females. Defining the mechanistic basis for reduced (in females) or increased (in males) AAA formation and progression may uncover potential therapeutic targets. The majority of studies examining sexual dimorphism focus on the role of sex hormones. However, genes residing on sex chromosomes, in addition to sex hormones, may contribute to sexual dimorphism of AAAs. For example, the X chromosome contains about 5% of the whole genome, but the role of sex chromosomes genes to sexual dimorphism of cardiovascular diseases such as AAAs is largely unknown. The purpose of this study was to determine the role of sex chromosomes as mediators of sex differences for angiotensin II (AngII)-induced AAAs in hypercholesterolemic mice. We used the four core genotype murine model, which enables the creation of phenotypically normal male and female mice with an XX versus XY sex chromosome complement, to test the hypothesis that an XY sex chromosome complement promotes AngII-induced AAAs. Transgenic male mice expressing the Sry gene on an autosome, but not on the Y-chromosome, were bred to female low-density lipoprotein receptor deficient mice to create male and female mice with an XX or an XY sex chromosome complement. In females, an XY sex chromosome complement doubled the incidence and markedly increased the severity of AngII-induced AAAs. To define mechanisms, we examined gene expression patterns in abdominal aortas and demonstrated elevated expression of inflammatory genes that were linked to increased MMP activity and oxidative stress in aortas from XY females. Moreover, administration of testosterone to XY females, to mimic males, resulted in a striking level of aneurysm rupture. In males, transcriptional profiling of abdominal aortas revealed 450 genes that were influenced by sex chromosomes. Infusion of AngII to XY males resulted in diffuse pathology along the length of the aorta, while XX males developed focal AAAs, with pathology reduced by orchiectomy in both genotypes. Thoracic aortas of XY males exhibited adventitial thickening which was not exist in thoracic aortas from XX males. Following a prolonged period (3 months) of AngII infusions XY males had AAAs with expanded aortic walls, while XX males had thin walled dilated AAAs. In summary, our findings demonstrate a remarkable effect of sex chromosome complement to regulate aortic vasculature and disease development. Aside from demonstrating mechanisms of sexual dimorphism of aortic diseases, these findings indicate that chronic sex hormone therapy in the aging and transgender population may have cardiovascular ramifications. Moreover, identification of targets influenced by sex chromosomes and/or sex hormones in a manner that predicts disease development may identify sex-specific approaches to cardiovascular therapy.

Background: Both human leukocyte antigen donor specific antibodies (HLA DSA) and non-HLA autoantibodies have been implicated in antibody-mediated rejection (AMR), allograft dysfunction, and allograft failure in kidney transplantation. Angiotensin II type 1 receptor antibody (AT1R-Ab), is a non-HLA antibody implicated in poor renal allograft outcomes, although its actions may be mediated through a different mechanistic pathway than HLA DSA. Objective: Our aim was to examine serum cytokine profiles associated with AT1R-Ab and distinguish them from those associated with HLA DSA in serially collected blood samples from a cohort of pediatric renal transplant recipients. Methods: 65 pediatric kidney transplant patients were monitored for 2 years post-transplant. Blood samples from early post-transplant and at 6, 12, and 24 months post-transplant and during suspected episodes of kidney transplant rejection were tested for AT1R-Ab, HLA DSA, and a panel of 6 cytokines (TNF-α, IFN-γ, IL-8, IL-1β, IL-6, and IL-17). Associations between antibodies and cytokines were evaluated. Results: AT1R-Ab, but not HLA DSA, was associated with elevations in TNF-α, IFN-γ, IL-8, IL-1β, IL-6, and IL-17. This relationship remained significant even when controlling for relevant clinical factors and potential confounders, and was consistent across time points. Conclusion: In contrast to HLA DSA, AT1R-Ab was associated with elevations in vascular inflammatory cytokines in the first 2 years post-transplant. This profile of vascular cytokines may be informative for designing further studies to understand the distinct pathophysiology of AT1R-Ab mediated allograft injury in kidney transplantation.

Regulatory variants, less well characterized than coding polymorphisms, have the potential to serve as clinically relevant biomarkers for determining drug efficacy or toxicity. This study focuses on the identification of functional regulatory single nucleotide polymorphisms (SNPs) in the candidate genes cytochrome P450 2C19 (CYP2C19), carboxylesterase 1A1 (CES1A1) and angiotensin-I converting enzyme (ACE), which are involved in the metabolism of many cardiovascular drugs. Using allelic mRNA ratios (an indicator of functional regulatory variants), classic molecular genetics and next-generation sequencing technologies, I demonstrate that cis-acting regulatory polymorphisms in these genes are present and may have utility as pharmacogenetic biomarkers. The drug metabolizing enzyme cytochrome P450 2C19 plays a major role in activation of clopidogrel (Plavix). Ultra-rapid metabolizer phenotype status has been associated with enhanced transcription caused by promoter allele CYP2C19*17. Here, I characterize the effect of CYP2C19*17 on gene expression in human liver. CYP2C19*17 heterozygotes and homozygotes show an increase in total CYP2C19 mRNA expression of 1.8-fold (p=0.028) and 2.9-fold (p=0.006), respectively compared to homozygous reference allele livers. Allelic mRNA ratios of ~1.8 fold (SD±0.6, p

Abstract: An increasing life expectancy in Canada has resulted in increasing proportions of elderly patients requiring anesthetic care for surgical procedures. Aging is associated with cardiovascular changes resulting in increased vasoconstriction and hypertension, and medications such as angiotensin converting enzyme (ACE) inhibitors are commonly prescribed for this reason. Propofol, a commonly used general anesthetic agent, is known to cause hypotension that can be exaggerated in the elderly population. Propofol’s influence on vascular reactivity has not been investigated in the aging vasculature. Moreover, patients using ACE inhibitors in the perioperative period may exhibit refractory hypotension when given a general anesthetic. Whether propofol differentially alters vascular function in arteries exposed to chronic ACE inhibition is also not known. Therefore, this thesis focused on 1) determining if there is a difference in vasodilating ability between the young and aging vasculature when exposed to propofol, and 2) determining if chronic ACE inhibition differentially alters vasodilation in the presence of propofol in the aging vasculature. Additionally, the utility of Intralipid as a potential treatment for propofol-induced hypotension was investigated in this thesis. Previous publications suggest that multiple mediators are involved in propofol-induced vascular relaxation. Here, the focus was on nitric oxide (NO) given the age-dependent decrease in NO bioavailability and based on studies documenting the contribution of NO following propofol administration in young mesenteric arteries, the primary vascular bed studied here. The experiments presented in this thesis demonstrate enhanced bioavailability of NO in the aging vasculature in the presence of propofol. Although NO is important, the aging vasculature chronically treated with ACE inhibitors possesses an enhanced vasodilation in the presence of propofol that results from a non-NO source. Finally, Intralipid is able to reverse propofol-induced vasodilation and hypotension in aging animals, demonstrating its potential to be utilized as a hemodynamic agent following propofol use. In conclusion, the results presented in this thesis are an important contribution to understanding the effects of propofol, a widely used general anesthetic agent, on the aging vasculature. This is of particular importance given the increasing proportion of the aging population presenting for surgery and requiring the care of the Anesthesiologist.

The renin-angiotensin system (RAS) is best-known as an endocrine system that regulates hydromineral balance and cardiovascular function. More recently, it has become evident that the RAS acts in an autocrine or paracrine fashion within many tissues to influence obesity and its comorbidities. The goal of these studies was to test specific hypotheses relating to how angiotensin-II (Ang-II; an end-product of the RAS) influences energy and glucose homeostasis. The overall hypothesis is that Ang-II acts in the brain to promote negative energy balance and peripherally to enhance energy storage. To assess the role of the RAS in energy and glucose homeostasis, body weight and composition, food intake and glucose tolerance were examined in rats given captopril. Captopril is an angiotensin-converting enzyme (ACE) inhibitor that prevents circulating Ang-II from being formed but does not readily access the brain. Rats fed either high-fat or low-fat diet and given captopril weighed less, had less body fat, and had improved glucose tolerance relative to controls. Rats given captopril also ate significantly less than free-fed controls, and comparisons to pair-fed controls indicated that the reduced weight gain and adiposity and improved glucose tolerance were due primarily to decreased food intake. Because systemic captopril elevates plasma and consequently brain angiotensin-I (Ang-I; the precursor for Ang-II and substrate for ACE), but does not itself enter the brain, we further hypothesized that still-active brain ACE would convert the increased Ang-I into Ang-II, and that the increased central Ang-II would contribute to systemic captopril-induced negative energy balance. Consistent with this, the reduction in food intake elicited by peripheral captopril was reversed by co-administration of the ACE inhibitor into the brain. These results suggest that captopril protects against diet-induced obesity, in part by elevating central Ang-II levels. These studies were extended by directly evaluating the role of RAS in the CNS regulation of energy balance. Osmotic minipumps were used to chronically administer Ang-II to rats in order to examine the effect of increased brain Ang-II signaling on energy balance. Chronic elevation of central Ang-II signaling resulted in reduced food intake, body weight gain and adiposity. The decrease in body weight and adiposity occurred relative to free-fed and pair-fed controls, implying that reduced food intake, in and of itself, does not underlie all of these effects. Consistent with this, rats administered Ang-II exhibited increased energy expenditure and enhanced expression of indices of adipose tissue sympathetic activation. Moreover, chronic icv Ang-II increased the anorectic corticotrophin-releasing and thyroid-releasing hormones within the hypothalamus. This hypothalamic gene expression profile coupled with the abundant angiotensin type-1 receptor expression within the paraventricular nucleus of the hypothalamus (PVN) led to the hypothesis that the PVN angiotensin type-1 receptor population may contribute to some of these processes. This hypothesis is discussed in the final chapter. Collectively, the experiments included in this dissertation support the overall hypothesis that Ang-II acts within the CNS to promote negative energy balance, and suggest that some contributing mechanisms include reduced food intake, elevated energy expenditure and enhanced sympathetic activation of adipose tissue.

Abstract: The renin-angiotensin system (RAS) has been implicated in vascular inflammation and atherosclerosis. Angiotensin II via the ATR1 can activate monocytes to produce inflammatory factors and increase adhesion. ATR1 expression is partly regulated by alternate splicing of the ATR1 gene. The RAS may also regulate immune function as part of the stress response: a model is proposed. ATR1 expression in two monocyte cell lines (U937 and THP-1) compared to a human microvascular endothelial cell line (HMEC1) was investigated. Western blot showed ATR1 protein expression in all cell types. PCR protocols targeted to the terminal protein-coding exon common to all transcript variants confirmed mRNA expression of the ATR1 gene in EC and monocytes. The 5 known splice variants were not identified in monocytes. 5’-RLM RACE was used to identify the 5’ untranslated ATR1 exons in monocytes. These data suggest a novel monocyte-specific splice variant, which may function in the cardiovascular disease process.

The renin angiotensin system (RAS) has been implicated in a variety of adipose tissue functions including tissue growth, differentiation, metabolism, and inflammation. While expression of all components necessary for a locally derived adipose tissue RAS have been demonstrated within adipose tissue, independence of local adipose RAS component concentrations from corresponding plasma RAS fluctuations has not been addressed. To analyze this, we varied in vivo rat plasma concentrations of two RAS components, renin and angiotensinogen (AGT), to determine the influence of their plasma concentrations on adipose and cardiac tissue levels in both perfused (plasma removed) and nonperfused samples. Variation of plasma RAS components was accomplished by 4 treatment groups: Normal, DOCA-salt, Bilateral nephrectomy, and Losartan. Adipose and cardiac tissue AGT concentrations correlated positively with plasma values. Perfusion of adipose tissue decreased AGT concentrations by 11.1% indicating that adipose tissue AGT was in equilibrium with plasma. Cardiac tissue renin levels positively correlated with plasma renin concentration for all treatments. In contrast, adipose tissue renin levels did not correlate with plasma renin, with the exception of extremely high plasma renin concentrations achieved in the Losartan treated group. These results suggest that adipose tissue may control its own local renin concentration independently of plasma renin as a potential mechanism for maintaining a functional local adipose RAS. Whereas adipose tissue possesses a local renin-angiotensin system, the synthesis and regulated release of renin has not been addressed. To that end we utilized differentiating 3T3-L1 cells and analyzed renin expression and secretion. Renin mRNA expression and protein enzymatic activity were not detectable in preadipocytes. However, upon differentiation, renin mRNA and both intracellular and extracellular renin activity were up regulated. In differentiated adipocytes, forskolin treatment resulted in a 28-fold increase in renin mRNA while TNF alpha treatment decreased renin mRNA 4-fold. IL-6, insulin, and angiotensin II (Ang II) were without effect. In contrast, forskolin and TNF alpha each increased renin protein secretion by 12- and 7-fold, respectively. Although both forskolin and TNFalpha induce lipolysis in adipocytes, fatty acids, prostaglandin E2 or lipopolysaccharide had no effect on renin mRNA or secretion. To evaluate mechanism(s) by which forskolin and/or TNF alpha are able to regulate renin secretion, a general lipase inhibitor (E600) and PKA inhibitor (H89) were used. Both inhibitors attenuated forskolin induced renin release while having no effect on TNF alpha regulated secretion. In contrast, E600 potentiated forskolin-stimulated renin mRNA levels while H89 had no effect. Neither inhibitor had any influence on TNFalpha regulation of renin mRNA. Relative to lean controls, renin expression was reduced 78% in the epididymal adipose tissue of obese male C57Bl/6J mice, consistent with TNF alpha-mediated down regulation of renin mRNA in the culture system. In conclusion, the expression and secretion of renin are regulated under a complex series of hormonal and metabolic determinants in mature 3T3-L1 adipocytes.

The median preoptic nucleus (MnPO) receives dense reciprocal inputs from both the subfornical organ (SFO) and organum vasculosum of the lamina terminalis (OVLT), the circumventricular organs known to be important as central neural sensors of circulating angiotensin II (ANG II). This thesis proposes to establish the role of the MnPO in chronic regulation of blood pressure based on the central hypothesis that the MnPO is a crucial component of the central sympathoexcitatory circuitry necessary for chronic blood pressure control following ANG II activation of the SFO and OVLT. Throughout the studies, cardiovascular responses to either pharmacological or physiological changes of circulating ANG II activity were compared between MnPO lesioned rats and sham lesioned controls. The first specific aim was designed to test the hypothesis that the intact MnPO is necessary for the full hypertensive response to chronic intravenous ANG II administration. In this specific aim, rats with electrolytic lesion of the MnPO displayed significantly attenuated hypertensive responses by day 7 through day 10 of ANG II infusion compared to sham lesioned rats. Therefore, we concluded that the MnPO is a crucial component of the central neuronal circuitry mediating chronic ANG II-induced hypertension. Sub-aim 1A was designed to determine the specific role of the MnPO neurons versus fibers of passage in the attenuated hypertensive responses to ANG II observed in the MnPO lesioned rats. In line with the findings of specific aim 1, rats with ibotenic acid lesion of the MnPO demonstrated attenuated responses to the hypertensive effect of chronic ANG II administration. However, the attenuated responses were less extensive relative to those seen in the electrolytic lesioned rats. Therefore, it was concluded that neuronal cell bodies in the MnPO are necessary for the full hypertensive response to chronic ANG II administration; however, damage of the fibers of passage partly contributes to the attenuated hypertensive responses observed in the electrolytic lesioned rats. The second specific aim was to determine the role of the MnPO in mediating the chronic hypotensive effect of the AT1 receptor antagonist, losartan. In this specific aim, rats with ibotenic acid lesion of the MnPO showed exaggerated hypotensive responses to chronic losartan administration. These findings were accompanied with a greater decline in total peripheral resistance in the MnPO lesioned rats. Therefore, we concluded that MnPO neurons do not mediate the chronic hypotensive effect of losartan and that the MnPO is not necessary for basal blood pressure control by endogenous ANG II. However, the findings suggested that the MnPO neurons likely participate in baroreflex mechanisms buffering against losartan-induced hypotension. The last specific aim was to establish the role of the MnPO in normal blood pressure control during chronic high dietary salt intake. Although plasma sodium concentration and osmolality were raised significantly in rats with electrolytic lesion of the MnPO during high salt intake, their mean arterial pressure and heart rate were comparable with those of sham lesioned rats throughout the study. Therefore, we concluded that the MnPO is not necessary to maintain normal blood pressure during high dietary salt intake. However, MnPO lesioned rats displayed less renal sodium retention during high salt intake compared to sham lesioned rats suggesting the role of the MnPO in the central neurohumoral control of sympathetic outflow, in particular, renal sympathetic activity, during chronic high salt intake. In conclusion, overall, the findings in this dissertation provide important insights into the role of the MnPO in the chronic hypertension induced by ANG II. Furthermore, they provide additional evidence of the integrative role of the MnPO in chronic normal blood pressure control by circulating ANG II, plasma osmolality, and the baroreflex.

Kanakamedala, Keerthy M.S., Department of Pharmacology and Toxicology, Wright State University, 2007. Role of Angiotensin converting enzymes ACE and ACE2 in diabetes induced cardiovascular dysfunction Cardiovascular disease is a long term complication of diabetes, which remains a leading cause of mortality and morbidity. There is recent evidence for activation of the Renin angiotensin system (RAS) in diabetic animals and humans. Emerging evidence shows that the vasoconstrictor actions of Ang II may be opposed by formation of the vasodilator, Ang (1-7). There is limited data on blood pressure in murine models of type 2 diabetes. The aim is to study the role of angiotensin converting enzymes ACE and ACE2 in diabetes induced cardiovascular dysfunction using type 2 diabetic murine mouse models (db/db mice). Both db/db mice and their controls were implanted with carotid telemetric probes for chronic monitoring of MAP (Mean Arterial pressure), heart rate and activity. At 8-9 weeks age, mice showed hyperinsulinemia, hyperglycemia and increase in body weight compared to their lean controls while MAP was not altered. At an older age (14-15 weeks) there was a significantly increased BP in the db/db mice compared to controls. In young (8 weeks) normotensive mice there was a highly significant increase in plasma ACE activity in db/db mice compared to controls. In contrast there was increased ACE2 activity and decreased ACE activity in kidney in 8 weeks old db/db mice compared to controls. No significant difference between ACE and ACE2 activity was observed in lungs and brain. In addition western blot analysis for ACE/ACE2 protein expression also revealed that there was a significant increase in kidney ACE2 and decrease in kidney ACE protein expression in 8 weeks old db/db mice compared to their lean controls. In addition no difference was observed between lung ACE and ACE2 protein expression. Increased plasma and kidney ACE activity and a decreased kidney ACE2 activity were observed in 24 weeks old db/db mice. In conclusion: 1. Eight weeks’ db/db mice were normotensive despite an increase in plasma ACE activity. 2. There was an age dependent increase in the BP but no change in heart rate (HR) in both young and old mice. 3. ACE2 may play a compensatory mechanism against development of hypertension in db/db mice in the early stage of type2 diabetes. 4. The up regulation of ACE coupled with a down regulation of ACE2 might be the cause for hypertension at a later stage in this murine model of type2 diabetes.

BACKGROUND: In hypertensive left ventricular hypertrophy (LVH), myocardial texture is altered by a disproportionate increase in fibrosis, but there is insufficient clinical evidence whether antihypertensive therapy or individual agents can induce regression of myocardial fibrosis. METHODS AND RESULTS: We compared the effects of an angiotensin II receptor antagonist with a beta-blocker on myocardial collagen volume (assessed by echoreflectivity and serum collagen markers) in 219 hypertensive patients with echocardiographically documented LVH. Patients were allocated randomly to receive losartan 50 to 100 mg/d (n=111) or atenolol 50 to 100 mg/d (n=99) with or without hydrochlorothiazide 12.5 to 25 mg/d for 36 weeks. Echoreflectivity analysis was conducted on ultrasound tracings of the midapex septum with specifically designed and validated software. A color histogram of reflecting echoes was obtained, and its spread (broadband [BB], previously shown to correlate directly with collagen volume fraction on endomyocardial biopsies) was used as the primary outcome measure. Mean color scale and serum markers of collagen synthesis (PIP, PIIIP) or degradation (CITP) were secondary outcome variables. Echoreflectivity analysis proved feasible in 106 patients (losartan 52, atenolol 54). Losartan reduced BB over 36 weeks (from 114.5 to 104.3 color levels, P

OBJECTIVES: We have investigated whether the A1166C polymorphism of the angiotensin II type 1 (AT1) receptor gene modulates the effects of angiotensin II on collagen type I turnover and myocardial stiffness in hypertension. METHODS: We studied 255 hypertensive patients before and after 1 year of treatment with either losartan (n = 185) or atenolol (n = 70). Serum concentrations of the carboxy-terminal propeptide of procollagen type I (PIP) and the carboxy-terminal telopeptide of collagen type I (CITP), markers of extracellular collagen type I synthesis and degradation, respectively, were measured by specific radioimmunoassays. Left ventricular chamber stiffness (KLV), was determined from the deceleration time of the early mitral filling wave, as measured by Doppler echocardiography. Hypertensives were genotyped by polymerase chain reaction and divided in two subgroups: AA (n = 126) and AC/CC (n = 129). RESULTS: Baseline PIP and KLV were increased (P < 0.01) in AA hypertensives compared with AC/CC hypertensives. No changes in baseline CITP were observed between the two subgroups of hypertensives. Confounding factors were similar between the two subgroups of hypertensives. Administration of losartan was associated with reduction (P < 0.01) in PIP and KLV in AA hypertensives but not in AC/CC hypertensives. Treatment with atenolol did not change PIP and KLV in either subgroup of hypertensives. No changes in CITP were observed with the two treatments. CONCLUSION: These findings suggest that the A1166C polymorphism of the AT1 receptor gene is associated with collagen type I synthesis and myocardial stiffness in patients with hypertensive heart disease.

Peptide transporters participate in the absorption of peptidomimetic drugs from the intestine and their reabsorption in the kidney. The focus of this research was to examine how peptide transporters interact with peptide-like ACE inhibitor drugs via (1) inhibition studies of radiolabeled GlySar uptake and (2) electrogenic studies using a two-microelectrode voltage-clamp technique in Xenopus oocytes expressing rat PEPT1 and PEPT2. It was found that ACE inhibitors displayed low-affinity interactions with both transporters. Kinetic analyses also demonstrated that quinapril inhibited the uptake of GlySar in a noncompetitive manner, while enalapril inhibited GlySar uptake in a competitive manner. Captopril and enalapril, but not quinapril, induced inwardly-directed currents in oocytes expressing rat PEPT2 as well as PEPT1. Quinapril was not a viable substrate but an inhibitor capable of reducing the captopril-evoked currents in oocytes. This finding is novel in that it argues against a free cc-amino group as being an absolute requirement for substrate recognition by PEPT2. Our findings also point to differences between PEPT1 and PEPT2 in their affinity to, rather than in their specificity for, ACE inhibitors. Additionally, the transport of captopril, enalapril and GlySar was studied in rat, rabbit and human PEPT1 and PEPT2 (expressed in oocytes) via an electrophysiological approach. An electrogenic and concentration-dependent transport of GlySar, enalapril and captopril was detected in all isoforms tested, which points to a unified peptide transport mechanism in mammals. Finally, using simultaneous radioactive uptake and current measurements (under voltage-clamp conditions), the stoichiometry of PEPT2 was determined to be 2:1 for protons:substrate.

It has been suggested that left ventricular fibrosis in spontaneously hypertensive rats (SHR) is the result of both exaggerated collagen synthesis and insufficient collagen degradation. We have shown previously that chronic treatment with the angiotensin II type 1 receptor antagonist losartan results in diminished synthesis of collagen type I molecules and reversal of myocardial fibrosis in SHR. This study was designed to investigate whether losartan also affects the extracellular degradation of collagen type I fibers in the left ventricle of SHR. The study was performed in 30-week-old normotensive Wistar-Kyoto rats (WKY), untreated SHR, and SHR treated with orally administered losartan (20 mg/kg per day) for 14 weeks before they were killed. Ventricular collagenase activity was determined by degradation of [(14)C]collagen with tissue extracts. Ventricular expression of tissue inhibitor of metalloproteinases 1 (TIMP-1) mRNA was analyzed by Northern blot. A histomorphometric study of the left ventricle was performed in all rats. Compared with WKY, SHR exhibited left ventricular hypertrophy, increased (P

This dissertation examines the interaction between angiotensin converting enzyme (ACE) inhibitors and renal oligopeptide transporters. Three types of experiments were performed using brush border membrane vesicles (BBMV) isolated from different sections of rabbit kidney. They include: (i) interaction of ACE inhibitors with high-affinity peptide transporter in kidney, and the relationship between inhibition potency and lipophilicity, (ii) competitive inhibition of GlySar uptake by enalapril in rabbit renal BBMV, and the effect on high-affinity peptide transporter, and (iii) GlySar uptake in rabbit renal BBMV isolated from outer-cortex or outer-medulla, and the evidence for heterogeneous distribution of oligopeptide transporters. Overall, ACE inhibitors showed different inhibition potencies toward the uptake of GlySar (15 $\mu$M). Only fosinopril and zofenopril showed IC50 values of less than 100 $\mu$M (55 $\mu$M and 81 $\mu$M, respectively). Other ACE inhibitors showed low-affinity interactions with the renal peptide transporter of which enalaprilat's interaction was the weakest (IC50 52 mM) toward GlySar uptake. With respect to structure-function relationships, the log IC50 and log distribution coefficient are significantly and linearly correlated. The IC50 decreases as the distribution coefficient increases. Subsequent studies regarding the interaction mechanism of ACE inhibitors with the renal high-affinity peptide transporter were performed using enalapril as a model ACE inhibitor. It was found that enalapril competitively inhibited the uptake of GlySar, a dipeptide substrate for peptide transporters, suggesting that enalapril might be a substrate for the high-affinity renal peptide transporter. In order to have a better understanding of the physiological and phannacological significance of the interaction between peptides or peptidomimetics (e.g., ACE inhibitors) and the renal peptide transporter, the intrarenal localization of PEPT1 and PEPT2 was assessed by determining GlySar uptake in BBMV isolated from outer-cortex (OC) or outer-medulla (OM) of rabbit kidneys. In contrast to earlier predictions, the high-affinity/low-capacity carrier system was found in both OC and OM membrane preparations (i.e., proximal convoluted and proximal straight tubules) while the low-affinity/high-capacity carrier system was found only in OC preparations (i.e., proximal convoluted tubules). In conclusion, although most ACE inhibitors appear to be weak inhibitors of GlySar uptake by the renal high-affinity peptide transporter, their inhibition potency increased as their lipophilicity increased. In addition, the competitive inhibition data suggest that enalapril may be a transportable substrate of the renal high-affinity peptide transporter. Finally, it appears that peptides and peptidomimetics are handled in a sequential manner in the kidney, first mainly by the low-affinity/high-capacity system (PEPT1) and then by the high-affinity/low-capacity transport system (PEPT2).