Erzsébet Lizanecz

Tissue-Specific Regulation of Microvascular Diameter: Opposite Functional Roles of Neuronal and Smooth Muscle Located Vanilloid Receptor-1

The transient receptor potential type V1 channel (vanilloid receptor 1, TRPV1) is a Ca2+-permeable nonspecific cation channel activated by various painful stimuli including ischemia. We hypothesized that TRPV1 is expressed in the arterioles and is involved in the regulation of microvascular tone. We found that TRPV1 stimulation by capsaicin (intra-arterial administration) of the isolated, perfused right hind limb of the rat increased vascular resistance (by 98 ± 21 mm Hg at 10 μg) in association with decreased skeletal muscle perfusion and elevation of skin perfusion (detected by dual-channel laser Doppler flowmetry). Denervation of the hind limb did not affect capsaicin-evoked changes in vascular resistance and tissue perfusion in the hind limb but reduced the elevation of perfusion in the skin. In isolated, pressurized skeletal (musculus gracilis) muscle arterioles (diameter, 147 ± 35 μm), capsaicin had biphasic effects: at lower concentrations, capsaicin (up to 10 nM) evoked dilations (maximum, 32 ± 13%), whereas higher concentrations (0.1-1 μM) elicited substantial constrictions (maximum, 66 ± 7%). Endothelium removal or inhibition of nitric-oxide synthase abolished capsaicin-induced dilations but did not affect arteriolar constriction. Expression of TRPV1 was detected by reverse transcriptase-polymerase chain reaction in the aorta and in cultured rat aortic vascular smooth muscle cells (A7r5). Immunohistochemistry revealed expression primarily in the smooth muscle layers of the gracilis arteriole. These data demonstrate the functional expression of TRPV1 in vascular smooth muscle cells mediating vasoconstriction of the resistance arteries. Because of the dual effects of TRPV1 stimulation on the arteriolar diameter (dilation in skin, constriction in skeletal muscle), we propose that TRPV1 ligands represent drug candidates for tissue-specific modulation of blood distribution.

Structure‐activity relationships of vanilloid receptor agonists for arteriolar TRPV1

BACKGROUND AND PURPOSE The transient receptor potential vanilloid 1 (TRPV1) plays a role in the activation of sensory neurons by various painful stimuli and is a therapeutic target. However, functional TRPV1 that affect microvascular diameter are also expressed in peripheral arteries and we attempted to characterize this receptor.

New perspectives in the renin-angiotensin-aldosterone system (RAAS) IV: circulating ACE2 as a biomarker of systolic dysfunction in human hypertension and heart failure.

Background Growing evidence exists for soluble Angiotensin Converting Enzyme-2 (sACE2) as a biomarker in definitive heart failure (HF), but there is little information about changes in sACE2 activity in hypertension with imminent heart failure and in reverse remodeling. Methods, Findings Patients with systolic HF (NYHAII-IV, enrolled for cardiac resynchronisation therapy, CRT, n = 100) were compared to hypertensive patients (n = 239) and to a healthy cohort (n = 45) with preserved ejection fraction (EF>50%) in a single center prospective clinical study. The status of the heart failure patients were checked before and after CRT. Biochemical (ACE and sACE2 activity, ACE concentration) and echocardiographic parameters (EF, left ventricular end-diastolic (EDD) and end-systolic diameter (ESD) and dP/dt) were measured. sACE2 activity negatively correlated with EF and positively with ESD and EDD in all patients populations, while it was independent in the healthy cohort. sACE2 activity was already increased in the hypertensive group, where signs for imminent heart failure (slightly decreased EF and barely increased NT-proBNP levels) were detected. sACE2 activities further increased in patients with definitive heart failure (EF<50%), while sACE2 activities decreased with the improvement of the heart failure after CRT (reverse remodeling). Serum angiotensin converting enzyme (ACE) concentrations were lower in the diseased populations, but did not show a strong correlation with the echocardiographic parameters. Conclusions Soluble ACE2 activity appears to be biomarker in heart failure, and in hypertension, where heart failure may be imminent. Our data suggest that sACE2 is involved in the pathomechanism of hypertension and HF.

Vascular metabolism of anandamide to arachidonic acid affects myogenic constriction in response to intraluminal pressure elevation

AIMS We hypothesized that arachidonic acid produced by anandamide breakdown contributes to the vascular effects of anandamide. MAIN METHODS Isolated, pressurized rat skeletal muscle arteries, which possess spontaneous myogenic tone, were treated with anandamide, arachidonic acid, capsaicin (vanilloid receptor agonist), WIN 55-212-2 (cannabinoid receptor agonist), URB-597 (FAAH inhibitor), baicalein (lipoxygenase inhibitor), PPOH (cytochrome P450 inhibitor), and indomethacin (cyclooxygenase inhibitor). Changes in the arteriolar diameter in response to the various treatments were measured. To assess the effect of anandamide metabolism, anandamide was applied for 20 min followed by washout for 40 min. This protocol was used to eliminate other, more direct effects of anandamide in order to reveal how anandamide metabolism may influence vasodilation. KEY FINDINGS Anandamide at a low dose (1μM) evoked a loss of myogenic tone, while a high dose (30 μM) not only attenuated the myogenic response but also evoked acute dilation. Both of these effects were inhibited by the FAAH inhibitor URB-597 and were mimicked by arachidonic acid. The CB1 and CB2 agonist R-WIN 55-212-2 and the vanilloid receptor agonist capsaicin were without effect on the myogenic response. The inhibition of the myogenic response by anandamide was blocked by indomethacin and PPOH, but not by baicalein or removal of the endothelium. FAAH expression in the smooth muscle cells of the blood vessels was confirmed by immunohistochemistry. SIGNIFICANCE Anandamide activates the arachidonic acid pathway in the microvasculature, affecting vascular autoregulation (myogenic response) and local perfusion.

Mistyping of angiotensinogen M235T alleles

Conflicting results are to be found in the literature on the relationship between the M235T polymorphism of the angiotensinogen (AGT) gene and hypertension. The controversy may be due to insufficient numbers of subjects, the variability of the inclusion criteria and the different genotype analysis methods used. We have experienced that the most frequently used, original polymerase chain reaction (PCR)−restriction fragment length polymorphism (RFLP) method involves significant uncertainties when the TT genotype is determined, independently of the restriction digestion. To make the determination more accurate, we improved the PCR by designing a new antisense primer containing only one mismatch instead of the two in the original protocol and also by adding DMSO to the PCR reaction mixture. The original and our improved methods were compared by using DNA from 123 patients: parallel determinations resulted in values of 33 MM, 90 MT and 0 TT with the original method and of 33 MM, 56 MT and 34 TT with the improved RFLP protocol. In summary, a plausible explanation for some of the conflicting data published on AGT M235T polymorphism may be that inaccuracies arose during the determination of the genotype.

Insertion/deletion polymorphism of the angiotensin-converting enzyme predicts left ventricular hypertrophy after renal transplantation.

BACKGROUND Kidney transplant recipients show a higher risk for cardiovascular complications, such as left ventricular hypertrophy and heart failure, leading to the premature death in many cases. METHODS We investigated the contribution of angiotensin-converting enzyme (ACE) insertion/deletion (I/D) polymorphism to the development of left ventricular hypertrophy (LVH), an indicator of heart disease progression among kidney transplant recipients. RESULTS We observed a significant correlation between graft function and left ventricular mass index. The occurrence of LVH or severe LVH was significantly greater among patients with at least one D-allele (ID or DD). CONCLUSION The use of ACE inhibitors or angiotensin receptor blockers seemed to be advantageous for patients with the ID and especially, the DD genotype.

Insertion/Deletion Polymorphism of Angiotensin-Converting Enzyme as a Risk Factor for Chronic Allograft Nephropathy

Angiotensin-converting enzyme (ACE) inhibitor therapy is widely used to treat chronic allograft nephropathy (CAN), which suggests a possible role of the renin-angiotensin system in the pathologic mechanism of the disease. The objective of this study was to investigate the possible link between CAN and ACE. The ACE insertion/deletion polymorphism and the amount and activity of ACE were determined in cadaver kidney recipients with CAN (n = 38) or normal renal function (n = 34). The DD genotype was observed significantly more frequently in the CAN group compared with the group with normal renal function. Moreover, the DD genotype was associated with a higher serum ACE concentration and greater serum ACE activity, compared with II genotype homozygotes. The insertion/deletion polymorphism of ACE affects ACE expression and activity in serum, and, therefore, may have an important role in the pathogenesis of CAN. These findings suggest that determination of the ACE genotype may be useful in identifying patients at high risk. In particular, the DD genotype may be considered an indication for ACE inhibitor therapy.

Circulating ACE2 activity correlates with cardiovascular disease development

It was shown recently that angiotensin-converting enzyme activity is limited by endogenous inhibition in vivo, highlighting the importance of angiotensin II (ACE2) elimination. The potential contribution of the ACE2 to cardiovascular disease progression was addressed. Serum ACE2 activities were measured in different clinical states (healthy, n=45; hypertensive, n=239; heart failure (HF) with reduced ejection fraction (HFrEF) n=141 and HF with preserved ejection fraction (HFpEF) n=47). ACE2 activity was significantly higher in hypertensive patients (24.8±0.8 U/ml) than that in healthy volunteers (16.2±0.8 U/ml, p=0.01). ACE2 activity further increased in HFrEF patients (43.9±2.1 U/ml, p=0.001) but not in HFpEF patients (24.6±1.9 U/ml) when compared with hypertensive patients. Serum ACE2 activity negatively correlated with left ventricular systolic function in HFrEF, but not in hypertensive, HFpEF or healthy populations. Serum ACE2 activity had a fair diagnostic value to differentiate HFpEF from HFrEF patients in this study. Serum ACE2 activity correlates with cardiovascular disease development: it increases when hypertension develops and further increases when the cardiovascular disease further progresses to systolic dysfunction, suggesting that ACE2 metabolism plays a role in these processes. In contrast, serum ACE2 activity does not change when hypertension progresses to HFpEF, suggesting a different pathomechanism for HFpEF, and proposing a biomarker-based identification of these HF forms.

Heteroduplex analysis using flow cytometric microbead assays to detect deletions, insertions, and single-strand lesions.

We explore the possibilities offered by flow cytometric microbead analysis to develop high throughput methods for the detection of deletions/insertions and single‐strand DNA lesions. The products of PCR reactions derived from reference and test samples are denatured and reannealed, then exposed to enzymatic or chemical treatments distinguishing homoduplices from heteroduplices. The biotin‐ and dye labeled reaction products are immobilized on microbeads and the homo‐ and heteroduplices are assessed in separate fluorescence channels, by flow cytometry. Using a model system based on the mixed lineage leukemia gene breakpoint cluster region, we demonstrate that deletions and insertions in genomic DNA can be detected, using S1 nuclease and chemical cleavage to distinguish hetero‐ from homoduplices, or a restriction enzyme cleaving only the homoduplices. Single‐strand discontinuities can also be detected, by combining nick‐translation, using labeled nucleotide, and flow cytometric microbead analysis. The methodical approaches demonstrated are applicable in a versatile manner in basic cell and molecular biological research and also promise direct application for high throughput screening of genetic diseases and lesions, including insertions or deletions of short sequence elements and single‐strand lesions formed at hypersensitive sites in response to apoptotic stimuli.