Method to develop a regional guide for the allergenic potential of tree pollen.

Allergic rhinitis, caused by airborne pollen, is a common disease with a great impact on the quality of life for patients and high costs for society. Prevention of high pollen concentrations in the air is relevant for creating a safe environment for allergic patients. Due to climate change, the heat in cities during the summer is a recurring problem. The local climate can be improved by using the cooling properties of trees, providing shade and cooling by evapotranspiration. When deciding which tree species will be planted, it is important to take into account the allergenicity of the pollen that the tree produces. Available guides, used all over the world, on the allergenicity of pollen are very divers in content and interpretation and not applicable for the Netherlands. In this study a method is described to develop a guide for the allergenic potential of tree pollen in a region, in this case the Netherlands. For the most common tree species in the Netherlands the scientific knowledge on the allergenicity of the pollen was collected, followed by an inventory on regional pollen abundance. Subsequently, the sensitization pattern in a patient group with possible inhalation allergy was analyzed. Based on these data allergenicity of the tree pollen was classified into five classes. Eight tree species/genera of the 61 most planted tree species in the Netherlands are considered to have a very strong to moderate allergenic potential. We propose to use this methodology to develop regional-specific guides classifying the allergenic potential of tree pollen.

Endothelium-derived hyperpolarizing factor and protein kinase G Iα activation: H2O2 versus S-nitrosothiols.

Protein kinase G (PKG) Iα mediates the cyclic guanosine monophosphate-mediated vasodilatory effects induced by NO. Endothelium-derived hyperpolarizing factors (EDHFs), like H2O2 can activate PKGIα in a cyclic guanosine monophosphate-independent manner, but whether this is true for all EDHFs (e.g., S-nitrosothiols) is unknown. Here, we investigated the contribution of PKGIα to bradykinin-, H2O2-, L-S-nitrosocysteine-, and light-induced relaxation in porcine coronary arteries, making use of the fact that thioredoxin reductase inhibition with auranofin or 1-chloro-2,4-dinitrobenzene potentiates PKGIα. Thioredoxin reductase inhibition potentiated bradykinin and H2O2, but not L-S-nitrosocysteine or light. The relaxations by the latter 2 and bradykinin, but not those by H2O2, were prevented by the soluble guanylyl cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. Yet, after S-nitrosothiol depletion with ethacrynic acid, thioredoxin reductase inhibition also potentiated light-induced relaxation, and this was prevented by the Na+-K+ ATPase inhibitor ouabain. This indicates that photorelaxation depends on sGC activation by S-nitrosothiols, while only after S-nitrosothiol depletion oxidized PKGIα comes into play, and acts via Na+-K+ ATPase. In conclusion, both bradykinin- and light-induced relaxation of porcine coronary arteries depend, at least partially, on oxidized PKGIα, and this does not involve sGC. H2O2 also acts via oxidized PKGIα in an sGC-independent manner. Yet, S-nitrosothiol-induced relaxation is PKGIα-independent. Clearly, PKG activation does not contribute universally to all EDHF responses, and targeting PKGIα may only mimick EDHF under certain conditions. It is therefore unlikely that PKGIα activators will be universal vasodilators.

Beneficial Effects of Combined AT1 Receptor/Neprilysin Inhibition (ARNI) Versus AT1 Receptor Blockade Alone in the Diabetic Eye.

Purpose: Dysfunction of the renin-angiotensin system (RAS) contributes to pathogenesis of diabetic retinopathy (DR). Yet RAS blockers have only limited beneficial effects on progression of DR in clinical trials. The natriuretic peptide system offsets RAS, so that enhancing the activity of this system on top of RAS blockade might be beneficial. Neprilysin has an important role in the degradation of natriuretic peptides. Therefore, we hypothesize that dual angiotensin receptor-neprilysin inhibition (ARNI) may outperform angiotensin receptor blocker (ARB) in protection against DR. We tested this hypothesis in streptozotocin-induced diabetic transgenic (mRen2)27 rats. Methods: Adult male diabetic (mRen2)27 rats were followed for 5 or 12 weeks. Treatment with vehicle, irbesartan (ARB), or ARB combined with the neprilysin inhibitor thiorphan (irbesartan+thiorphan [ARNI]) occurred during the final 3 weeks. Retinal cell death, gliosis, and capillary loss were evaluated. Real-time polymerase chain reaction (RT-PCR) analyses were performed to quantify the retinal level of inflammatory cell markers. Results: Both ARB- and ARNI-treated groups showed similarly reduced retinal apoptotic cell death, gliosis, and capillary loss compared to the vehicle-treated group in the 5-week study. Treatment with ARNI reduced the expression of inflammatory markers more than ARB treatment in the 5-week study. In the 12-week study, ARNI treatment showed significantly more reduction in apoptotic cell death (51% vs. 25% reduction), and capillary loss (68% vs. 43% reduction) than ARB treatment. Conclusions: Treatment with ARNI provides better protection against DR in diabetic (mRen2)27 transgenic rats, compared to ARB alone. This approach may be a promising treatment option for patients with DR.

Blood pressure-independent renoprotection in diabetic rats treated with AT1 receptor-neprilysin inhibition compared with AT1 receptor blockade alone.

ARNI [dual AT1 (angiotensin II type 1) receptor-neprilysin inhibition] exerts beneficial effects on blood pressure and kidney function in heart failure, compared with ARB (AT1 receptor blockade) alone. We hypothesized that ARNI improves cardiac and kidney parameters in diabetic TGR(mREN2)27 rats, an angiotensin II-dependent hypertension model. Rats were made diabetic with streptozotocin for 5 or 12 weeks. In the final 3 weeks, rats were treated with vehicle, irbesartan (ARB) or irbesartan+thiorphan (ARNI). Blood pressure, measured by telemetry in the 5-week group, was lowered identically by ARB and ARNI. The heart weight/tibia length ratio in 12-week diabetic animals was lower after ARNI compared with after ARB. Proteinuria and albuminuria were observed from 8 weeks of diabetes onwards. ARNI reduced proteinuria more strongly than ARB, and a similar trend was seen for albuminuria. Kidneys of ARNI-treated animals showed less severe segmental glomerulosclerosis than those of ARB-treated animals. After 12 weeks, no differences between ARNI- and ARB-treated animals were found regarding diuresis, natriuresis, plasma endothelin-1, vascular reactivity (acetylcholine response) or kidney sodium transporters. Only ARNI-treated rats displayed endothelin type B receptor-mediated vasodilation. In conclusion, ARNI reduces proteinuria, glomerulosclerosis and heart weight in diabetic TGR(mREN2)27 rats more strongly than does ARB, and this occurs independently of blood pressure.

Optimum AT1 receptor-neprilysin inhibition has superior cardioprotective effects compared with AT1 receptor blockade alone in hypertensive rats.

Neprilysin inhibitors prevent the breakdown of bradykinin and natriuretic peptides, promoting vasodilation and natriuresis. However, they also increase angiotensin II and endothelin-1. Here we studied the effects of a low and a high dose of the neprilysin inhibitor thiorphan on top of AT1 receptor blockade with irbesartan versus vehicle in TGR(mREN2)27 rats with high renin hypertension. Mean arterial blood pressure was unaffected by vehicle or thiorphan alone. Irbesartan lowered blood pressure, but after 7 days pressure started to increase again. Low- but not high-dose thiorphan prevented this rise. Only during exposure to low-dose thiorphan plus irbesartan did heart weight/body weight ratio, cardiac atrial natriuretic peptide expression, and myocyte size decrease significantly. Circulating endothelin-1 was not affected by low-dose thiorphan with or without irbesartan, but increased after treatment with high-dose thiorphan plus irbesartan. This endothelin-1 rise was accompanied by an increase in renal sodium-hydrogen exchanger 3 protein abundance, and an upregulation of constrictor vascular endothelin type B receptors. Consequently, the endothelin type B receptor antagonist BQ788 no longer enhanced endothelin-1-induced vasoconstriction (indicative of endothelin type B receptor-mediated vasodilation), but prevented it. Thus, optimal neprilysin inhibitor dosing reveals additional cardioprotective effects on top of AT1 receptor blockade in renin-dependent hypertension.

Combined renin inhibition/(pro)renin receptor blockade in diabetic retinopathy--a study in transgenic (mREN2)27 rats.

Dysfunction of renin-angiotensin system (RAS) contributes to the pathogenesis of diabetic retinopathy (DR). Prorenin, the precursor of renin is highly elevated in ocular fluid of diabetic patients with proliferative retinopathy. Prorenin may exert local effects in the eye by binding to the so-called (pro)renin receptor ((P)RR). Here we investigated the combined effects of the renin inhibitor aliskiren and the putative (P)RR blocker handle-region peptide (HRP) on diabetic retinopathy in streptozotocin (STZ)-induced diabetic transgenic (mRen2)27 rats (a model with high plasma prorenin levels) as well as prorenin stimulated cytokine expression in cultured Müller cells. Adult (mRen2)27 rats were randomly divided into the following groups: (1) non-diabetic; (2) diabetic treated with vehicle; (3) diabetic treated with aliskiren (10 mg/kg per day); and (4) diabetic treated with aliskiren+HRP (1 mg/kg per day). Age-matched non-diabetic wildtype Sprague-Dawley rats were used as control. Drugs were administered by osmotic minipumps for three weeks. Transgenic (mRen2)27 rat retinas showed increased apoptotic cell death of both inner retinal neurons and photoreceptors, increased loss of capillaries, as well as increased expression of inflammatory cytokines. These pathological changes were further exacerbated by diabetes. Aliskiren treatment of diabetic (mRen2)27 rats prevented retinal gliosis, and reduced retinal apoptotic cell death, acellular capillaries and the expression of inflammatory cytokines. HRP on top of aliskiren did not provide additional protection. In cultured Müller cells, prorenin significantly increased the expression levels of IL-1α and TNF-α, and this was completely blocked by aliskiren or HRP, their combination, (P)RR siRNA and the AT1R blocker losartan, suggesting that these effects entirely depended on Ang II generation by (P)RR-bound prorenin. In conclusion, the lack of effect of HRP on top of aliskiren, and the Ang II-dependency of the ocular effects of prorenin in vitro, argue against the combined application of (P)RR blockade and renin inhibition in diabetic retinopathy.

Deterioration of kidney function by the (pro)renin receptor blocker handle region peptide in aliskiren-treated diabetic transgenic (mRen2)27 rats.

Dual renin-angiotensin system (RAS) blockade in diabetic nephropathy is no longer feasible because of the profit/side effect imbalance. (Pro)renin receptor [(P)RR] blockade with handle region peptide (HRP) has been reported to exert beneficial effects in various diabetic models in a RAS-independent manner. To what degree (P)RR blockade adds benefits on top of RAS blockade is still unknown. In the present study, we treated diabetic TGR(mREN2)27 rats, a well-established nephropathy model with high prorenin levels [allowing continuous (P)RR stimulation in vivo], with HRP on top of renin inhibition with aliskiren. Aliskiren alone lowered blood pressure and exerted renoprotective effects, as evidenced by reduced glomerulosclerosis, diuresis, proteinuria, albuminuria, and urinary aldosterone levels as well as diminished renal (P)RR and ANG II type 1 receptor expression. It also suppressed plasma and tissue RAS activity and suppressed cardiac atrial natriuretic peptide and brain natriuretic peptide expression. HRP, when given on top of aliskiren, did not alter the effects of renin inhibition on blood pressure, RAS activity, or aldosterone. However, it counteracted the beneficial effects of aliskiren in the kidney, induced hyperkalemia, and increased plasma plasminogen activator-inhibitor 1, renal cyclooxygenase-2, and cardiac collagen content. All these effects have been linked to (P)RR stimulation, suggesting that HRP might, in fact, act as a partial agonist. Therefore, the use of HRP on top of RAS blockade in diabetic nephropathy is not advisable.

Renin inhibitor VTP-27999 differs from aliskiren: focus on their intracellular accumulation and the (pro)renin receptor.

BACKGROUND: VTP-27999 is a renin inhibitor with an IC50 that is comparable to that of aliskiren, but with a higher bioavailability. Unexpectedly, VTP-27999, unlike aliskiren, did not unfold renin's precursor, prorenin, and increased the affinity of the antibodies applied in renin immunoassays. METHODS: Here we verified to what degree these differences affect intracellular renin inhibitor accumulation in renin-synthesizing human mast cells (HMC-1), and (pro)renin's signaling via the (pro)renin receptor ((P)RR) in rat vascular smooth muscle cells. We also addressed the consequences of (P)RR knockdown by small-interfering (si) RNA on (pro)renin release. Finally, making use of FRET(Bodipy-FL)-labeled aliskiren, we studied, by subcellular fractionation, the cellular distribution pattern of this renin inhibitor. RESULTS: VTP-27999 accumulated at higher levels in HMC-1 cells than aliskiren, allowing this inhibitor to block intracellular renin at approximately five-fold lower medium levels. Labeled aliskiren accumulated in mitochondria and lysosomes, and its distribution pattern was different from that of renin. Moreover, the intracellular accumulation of both inhibitors in nonrenin-synthesizing HEK293 cells was not different from that in HMC-1 cells, suggesting that it is renin synthesis-independent. VTP-27999, but not aliskiren, blocked renin's capacity to stimulate extracellular signal-regulated kinase 1/2 phosphorylation in vascular smooth muscle cells, whereas neither inhibitor interfered with prorenin-induced signaling. (P)RR knockdown greatly increased renin (and to a lesser degree, prorenin) release, without affecting the capacity of forskolin or cAMP to stimulate renin release. CONCLUSION: VTP-27999 differs from aliskiren regarding its level of intracellular accumulation and its capacity to interfere with renin signaling via the (P)RR, and the (P)RR determines prorenin-renin conversion and constitutive (but not regulated) (pro)renin release.

Long-lasting physiological antagonism of calcitonin gene-related peptide towards endothelin-1 in rat mesenteric arteries and human coronary arteries.

Endothelin-1 causes long-lasting contraction via endothelin type A receptor (ETAR) in isolated rat mesenteric arteries (RMA) that cannot be readily terminated by removing the agonist, or by adding the ETAR antagonist BQ123 or the NO donor sodium nitroprusside. It could be terminated by adding calcitonin-gene related peptide (CGRP), most likely because CGRP causes ET-1/ETAR dissociation. Here we investigated this phenomenon in human coronary microarteries (HCMA). We simultaneously verified the effects of CGRP in RMA and HCMA towards other vasoconstrictors, i.e., the α1-adrenoceptor agonist phenylephrine, the thromboxane A2 analog U46619 (9,11-dideoxy-11α,9α-epoxy-methano-prostaglandin F2α) and KCl. Long-lasting contraction (remaining after washing away the agonist) was observed for ET-1 in RMA, but not HCMA. Constrictions to phenylephrine, U46619 or KCl did not last upon washing. When added on top of ET-1-initiated contraction in RMA, CGRP effectively counteracted vasoconstriction, i.e., it caused full relaxation. Inhibitory effects of CGRP were also observed when briefly exposing RMA and HCMA to CGRP 1h before the addition of ET-1. Similar inhibitory effects of transient CGRP pre-incubation were seen towards phenylephrine, U46619 or KCl in RMA and HCMA. In conclusion, our data imply that CGRP, like ET-1, causes long-lasting effects that remain apparent up to 1h after its removal from the organ bath. Thus, in addition to the reported dissociation of ET-1/ETAR complexes, CGRP causes long-lasting non-selective arterial smooth muscle relaxation that may add to the neuropeptide being a physiological antagonist of arterial effects of ET-1. Long-lasting, washout-resistant ET-1/ETAR interaction does not occur in HCMAs.

The (pro)renin receptor blocker handle region peptide upregulates endothelium-derived contractile factors in aliskiren-treated diabetic transgenic (mREN2)27 rats.

BACKGROUND: Elevated prorenin levels associate with microvascular complications in patients with diabetes mellitus, possibly because prorenin affects vascular function in diabetes mellitus, for example by generating angiotensins following its binding to the (pro)renin receptor [(P)RR]. Here we evaluated whether the renin inhibitor aliskiren, with or without the putative (P)RR antagonist handle region peptide (HRP) improved the disturbed vascular function in diabetic TGR(mREN2)27 rats, a high-prorenin, high-(P)RR hypertensive model. METHODS: Telemetry transmitters were implanted to monitor blood pressure. After 3 weeks of treatment, rats were sacrificed, and iliac and mesenteric arteries were removed to evaluate vascular reactivity. RESULTS: Diabetes mellitus enhanced the contractile response to nitric oxide synthase (NOS) blockade, potentiated the response to phenylephrine, diminished the effectiveness of endothelin type A (ETA) receptor blockade and allowed acetylcholine to display constrictor, cyclo-oxygenase-2 mediated, endothelium-dependent responses in the presence of NOS inhibition and blockers of endothelium-derived hyperpolarizing factors. Aliskiren normalized blood pressure, suppressed renin activity, and reversed the above vascular effects, with the exception of the altered effectiveness of ETA receptor blockade. Remarkably, when adding HRP on top of aliskiren, its beneficial vascular effects either disappeared or were greatly diminished, although HRP did not alter the effect of aliskiren on blood pressure and renin activity. CONCLUSIONS: Renin inhibition improves vascular dysfunction in diabetic hypertensive rats, and HRP counteracts this effect independently of blood pressure and angiotensin. (P)RR blockade therefore is unlikely to be a new tool to further suppress the renin-angiotensin system (RAS) on top of existing RAS blockers.

Nitrite- and nitroxyl-induced relaxation in porcine coronary (micro-) arteries: underlying mechanisms and role as endothelium-derived hyperpolarizing factor(s).

To investigate the vasorelaxant efficacy of nitrite and nitroxyl (HNO) in porcine coronary (micro)arteries (PC(M)As), evaluating their role as endothelium-derived hyperpolarizing factors (EDHFs), preconstricted PCAs and PCMAs were exposed to UV light (a well-known inductor of nitrite; wave-length: 350-370nm), nitrite, the HNO donor Angeli's salt, or bradykinin. UV light-induced relaxation of PCAs increased identically after endothelium removal and endothelial nitric oxide (NO) synthase (eNOS) blockade. UV light-induced relaxation diminished during Na(+)-K(+)-ATPase inhibition and S-nitrosothiol-depletion, and disappeared during NO scavenging with hydroxocobalamin or soluble guanylyl cyclase (sGC) inhibition with ODQ. Nitrite-induced relaxation of PCAs required millimolar levels, i.e., >1000 times endogenous vascular nitrite. Angeli's salt relaxed PCMAs more potently than PCAs, and this was due to the fact that HNO directly activated sGC in PCMAs, whereas in PCAs this occurred following its conversion to NO only. sGC activation by NO/HNO resulted in Na(+)-K(+)-ATPase stimulation and K(v) channel activation. The HNO scavenger l-cysteine blocked bradykinin-induced relaxation in PCAs, and potentiated it in PCMAs. The latter did not occur in the presence of hydroxocobalamin, suggesting that it depended on l-cysteine-induced generation of vasorelaxant S-nitrosothiols. In all experimental setups, incubation with red wine extract mimicked the effects of ODQ. In conclusion, nitrite, via its conversion to NO and S-nitrosothiols, and HNO, either directly, or via its conversion to NO, mediate relaxant effects involving the sGC-cGMP pathway, Na(+)-K(+)-ATPase and/or K(v) channels. Red wine extract counteracts these beneficial effects. NO blocks nitrite activation, and HNO, but not nitrite, may act as EDHF in the coronary vascular bed.

Compound 21 induces vasorelaxation via an endothelium- and angiotensin II type 2 receptor-independent mechanism.

Angiotensin II type 2 (AT(2)) receptor stimulation has been linked to vasodilation. Yet, AT(2) receptor-independent hypertension and hypotension (or no effect on blood pressure) have been observed in vivo after application of the AT(2) receptor agonist compound 21 (C21). We, therefore, studied its effects in vitro, using preparations known to display AT(2) receptor-mediated responses. Hearts of Wistar rats, spontaneously hypertensive rats (SHRs), C57Bl/6 mice, and AT(2) receptor knockout mice were perfused according to Langendorff. Mesenteric and iliac arteries of these animals, as well as coronary microarteries from human donor hearts, were mounted in Mulvany myographs. In the coronary vascular bed of Wistar rats, C57Bl/6 mice, and AT(2) receptor knockout mice, C21 induced constriction followed by dilation. SHR hearts displayed enhanced constriction and no dilation. Irbesartan (angiotensin II type 1 receptor blocker) abolished the constriction and enhanced or (in SHRs) reintroduced dilation, and PD123319 (AT(2) receptor blocker) did not block the latter. C21 relaxed preconstricted vessels of all species, and this did not depend on angiotensin II receptors, the endothelium, or the NO-guanylyl cyclase-cGMP pathway. C21 constricted SHR iliac arteries but none of the other vessels, and irbesartan prevented this. C21 shifted the concentration-response curves to U46619 (thromboxane A(2) analog) and phenylephrine (α-adrenoceptor agonist) but not ionomycine (calcium ionophore) to the right. In conclusion, C21 did not cause AT(2) receptor-mediated vasodilation. Yet, it did induce vasodilation by blocking calcium transport into the cell and constriction via angiotensin II type 1 receptor stimulation. The latter effect is enhanced in SHRs. These data may explain the varying effects of C21 on blood pressure in vivo.

(Pro)renin and its receptors: pathophysiological implications.

Tissue angiotensin generation depends on the uptake of circulating (kidney-derived) renin and/or its precursor prorenin [together denoted as (pro)renin]. Since tissue renin levels are usually somewhat higher than expected based upon the amount of (renin-containing) blood in tissue, an active uptake mechanism has been proposed. Several candidates have been evaluated in the past three decades, including a renin-binding protein, the mannose 6-phosphate/insulin-like growth factor II receptor and the (pro)renin receptor. Although the latter seemed the most promising, its nanomolar affinity for renin and prorenin is several orders of magnitude above their actual (picomolar) levels in blood, raising doubt on whether (pro)renin-(pro)renin receptor interaction will ever occur in vivo. A wide range of in vitro studies have now demonstrated (pro)renin-receptor-induced effects at nanomolar renin and prorenin concentrations, resulting in a profibrotic phenotype. In addition, beneficial in vivo effects of the putative (pro)renin receptor blocker HRP (handle region peptide) have been observed, particularly in diabetic animal models. Despite these encouraging results, many other studies have reported either no or even contrasting effects of HRP, and (pro)renin-receptor-knockout studies revealed lethal consequences that are (pro)renin-independent, most probably due to the fact that the (pro)renin receptor co-localizes with vacuolar H+-ATPase and possibly determines the stability of this vital enzyme. The present review summarizes all of the recent findings on the (pro)renin receptor and its blockade, and critically compares it with the other candidates that have been proposed to mediate (pro)renin uptake from blood. It ends with the conclusion that the (pro)renin-(pro)renin receptor interaction, if it occurs in vivo, is limited to (pro)renin-synthesizing organs such as the kidney.

Angiotensin II-aldosterone interaction in human coronary microarteries involves GPR30, EGFR, and endothelial NO synthase.

AIMS: The aim of this study was to investigate the aldosterone-angiotensin (Ang) II interaction in human coronary microarteries (HCMAs). METHODS AND RESULTS: HCMAs, obtained from 75 heart-beating organ donors, were mounted in myographs and exposed to Ang II, either directly or following a 30-min pre-incubation with aldosterone, 17ß-oestradiol, hydrocortisone, the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580, the extracellular regulated kinase 1/2 (ERK1/2) inhibitor PD98059, the GPR30 antagonist G15, or the epidermal growth factor receptor (EGFR) antagonist AG1478. Ang II constricted HCMAs in a concentration-dependent manner. All steroids, at nanomolar levels, potentiated Ang II and G15 prevented this effect. The potentiation disappeared or was reversed into Ang II antagonism at micromolar steroid levels. NO synthase (NOS) inhibition prevented the latter antagonism in the case of 17ß-oestradiol, whereas both aldosterone and 17ß-oestradiol at micro- (but not nano-) molar levels induced endothelial NOS phosphorylation in human umbilical vein endothelial cells. AG1478, but not SB203580 or PD98059, abolished the Ang II-induced contraction in the presence of aldosterone or 17ß-oestradiol, and none of these drugs affected Ang II alone. CONCLUSION: Steroids including aldosterone affect Ang II-induced vasoconstriction in a biphasic manner. Potentiation occurs at nanomolar steroid levels and depends on GPR30 and EGFR transactivation. At micromolar steroid levels, this potentiation either disappears (aldosterone and hydrocortisone) or is reversed into an inhibition (17ß-oestradiol), and this is due to the endothelial NOS activation that occurs at such concentrations.

Renin- and prorenin-induced effects in rat vascular smooth muscle cells overexpressing the human (pro)renin receptor: does (pro)renin-(pro)renin receptor interaction actually occur?

Renin/prorenin binding to the (pro)renin receptor ([P]RR) results in direct (angiotensin-independent) second-messenger activation in vitro, whereas in vivo studies in rodents overexpressing prorenin (≈400-fold) or the (P)RR do not support such activation. To solve this discrepancy, DNA synthesis, extracellular signal-regulated kinase 1/2 phosphorylation, and plasminogen-activator inhibitor 1 release were evaluated in wild-type and human (P)RR-overexpressing vascular smooth muscle cells after their incubation with 1 to 80 nmol/L of (pro)renin. Human prorenin (4 nmol/L, ie, ≈800-fold above normal) + angiotensinogen increased DNA synthesis in human (P)RR cells only in an angiotensin II type 1 receptor-dependent manner. Prorenin at this concentration also increased plasminogen-activator inhibitor 1 release via angiotensin. Prorenin alone at 4 nmol/L was without effect, but at 20 nmol/L (≈4000-fold above normal) it activated extracellular signal-regulated kinase 1/2 directly (ie, independent of angiotensin). Renin at concentrations of 1 nmol/L (≈2000-fold above normal) and higher directly stimulated DNA synthesis, extracellular signal-regulated kinase 1/2 phosphorylation, and plasminogen-activator inhibitor 1 release in wild-type and human (P)RR cells, and similar effects were seen for rat renin, indicating that they were mediated via the rat (P)RR. In conclusion, angiotensin generation depending on prorenin-(P)RR interaction may occur in transgenic rodents overexpressing prorenin several 100-fold. Direct (pro)renin-induced effects via the (P)RR require agonist concentrations that are far above the levels in wild-type and transgenic rats. Therefore, only prorenin (and not [P]RR) overexpression will result in an angiotensin-dependent phenotype, and direct renin-(P)RR interaction is unlikely to ever occur in nonrenin-synthesizing organs.

Urinary renin, but not angiotensinogen or aldosterone, reflects the renal renin-angiotensin-aldosterone system activity and the efficacy of renin-angiotensin-aldosterone system blockade in the kidney.

OBJECTIVE: To study which renin-angiotensin-aldosterone system (RAAS) component best reflects renal RAAS activity. METHODS AND RESULTS: We measured urinary and plasma renin, prorenin, angiotensinogen, aldosterone, albumin and creatinine in 101 diabetic and nondiabetic patients with or without hypertension. Plasma prorenin was elevated in diabetic patients. Urinary prorenin was undetectable. Urinary albumin and renin were higher in diabetic patients. Men had higher plasma renin/prorenin levels, and lower plasma angiotensinogen levels than women. Plasma creatinine and albumin were also higher in men. Urinary RAAS components showed no sexual dimorphism, whereas urinary creatinine and albumin were higher in men. Angiotensin-converting enzyme inhibitors and angiotensin II type 1 receptor blockers increased plasma renin and decreased plasma angiotensinogen, without altering plasma aldosterone. In contrast, in urine, these drugs decreased renin and aldosterone without affecting angiotensinogen. When analyzing all patients together, urinary angiotensinogen excretion closely mimicked that of albumin, whereas urinary angiotensinogen and albumin levels both were 0.05% or less of their concomitant plasma levels. This may reflect the identical glomerular filtration and tubular handling of both proteins, which have a comparable molecular weight. In contrast, urinary renin excretion did not correlate with urinary albumin excretion, and the urinary/plasma concentration ratio of renin was more than 200 times the ratio of albumin, despite its comparable molecular weight. Urinary aldosterone excretion closely followed urinary creatinine excretion. CONCLUSION: The increased urinary renin levels in diabetes and the decreased urinary renin levels following RAAS blockade, occurring independently of changes in plasma renin, reflect the activated renal RAAS in diabetes and the success of RAAS blockade in the kidney, respectively. Urinary renin, therefore, more closely reflects renal RAAS activity than urinary angiotensinogen or aldosterone.

Specific coronary drug-eluting stents interfere with distal microvascular function after single stent implantation in pigs.

OBJECTIVES: The aim of this study was to compare the effects of single drug-eluting stents (DES) on porcine coronary function distal to the stent in vivo and in vitro. BACKGROUND: The mechanism of endothelial dysfunction occurring in human coronary conduit arteries up to 9 months after DES implantation is unknown. METHODS: A sirolimus-eluting stent (SES), paclitaxel-eluting stent (PES), and a bare-metal stent (BMS) were implanted in the 3 coronary arteries of 11 pigs. After 5 weeks, in vivo responses in distal coronary flow to different doses of bradykinin (BK) and nitrates were measured. In vitro, vasodilation to BK and nitrates, as well as vasoconstriction to endothelin (ET)-1 were assessed in both distal coronary conduit and small arteries. In addition, contributions of nitric oxide (NO) and endothelium-derived hyperpolarizing factors (EDHFs) and cyclic guanosine monophosphate (cGMP) responses to BK-stimulation were determined in vitro. RESULTS: Both DES did not alter in vivo distal vasomotion. In vitro distal conduit and small arterial responses to BK were also unaltered; DES did not alter the BK-induced increase in cGMP. However, after NO synthase blockade, PES showed a reduced BK-response in distal small arteries as compared with BMS and SES (p < 0.05). The ET-1-induced vasoconstriction and vascular smooth muscle cell function were unaltered. CONCLUSIONS: In this study of single stenting in healthy porcine coronaries for 5 weeks, SES did not affect distal coronary vascular function, whereas PES altered distal endothelial function of small arteries under conditions of reduced NO bioavailability. Therefore, specifically the EDHF component of microvascular function seems affected by PES.

Light-induced vs. bradykinin-induced relaxation of coronary arteries: do S-nitrosothiols act as endothelium-derived hyperpolarizing factors?

BACKGROUND: Light-induced relaxation depends on S-nitrosothiols. S-Nitrosothiols may also serve as endothelium-derived hyperpolarizing factors, mediating the relaxant response of porcine coronary arteries (PCAs) to bradykinin. Here we compared the mechanism of light-induced and bradykinin-induced PCA relaxation. METHODS: PCAs were mounted in organ baths in the dark, preconstricted and exposed to polychromatic light (5 min) or 100 nmol/l bradykinin. RESULTS: Light relaxed PCAs by maximally 71 +/- 1%. S-Nitrosothiol depletion abolished this relaxation. Relaxations diminished following repetitive light exposures, particularly if the dark periods between the light exposures were less than 10 min, and increased following endothelium removal or nitric oxide synthase blockade with N(omega)-nitro-L-arginine methyl ester (L-NAME), despite the prevention of guanosine-3',5'-cyclic monophosphate generation by the latter two procedures. Thus, reloading of the storage pools occurs in the dark, endothelial nitric oxide inhibits this process and photorelaxation does not depend on guanosine-3',5'-cyclic monophosphate. Bradykinin relaxed PCAs by 69 +/- 3%. The nitric oxide scavenger hydroxocobalamin and the Na+-K+ ATPase inhibitor ouabain abolished the responses to bradykinin and light. The guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one abolished the response to light, and, like L-NAME, blocked the response to bradykinin by more than 50%. On top of L-NAME, intermediate and small conductance Ca2+-dependent K+ channel (IKCa/SKCa) blockade further reduced the response to bradykinin and enhanced photorelaxation. CONCLUSION: Photorelaxation depends on stored S-nitrosothiols and their release/synthesis is negatively affected by endothelial nitric oxide and IKCa/SKCa. S-Nitrosothiols activate endothelial IKCa/SKCa and, via guanylyl cyclase, smooth muscle Na+-K+ ATPase. Thus, they possess all properties of a bradykinin-induced endothelium-derived hyperpolarizing factor.

Diabetic complications: a role for the prorenin-(pro)renin receptor-TGF-beta1 axis?

Morbidity and mortality of diabetes mellitus are strongly associated with cardiovascular disease including nephropathy. A discordant tissue renin-angiotensin system (RAS) might be a mediator of the endothelial dysfunction leading to both micro- and macrovascular complications of diabetes. The elevated plasma levels of prorenin in diabetic subjects with microvascular complications might be part of this discordant RAS, especially since the plasma renin levels in diabetes are low. Prorenin, previously thought of as an inactive precursor of renin, is now known to bind to a (pro)renin receptor, thus activating locally angiotensin-dependent and -independent pathways. In particular, the stimulation of the transforming growth factor-beta (TGF-beta) system by prorenin could be an important contributor to diabetic disease complications. This review discusses the concept of the prorenin-(pro)renin receptor-TGF-beta(1) axis, concluding that interference with this pathway might be a next logical step in the search for new therapeutic regimens to reduce diabetes-related morbidity and mortality.