Mechanism of pro-MMP9 activation in co-culture of pro-inflammatory macrophages and cardiomyocytes.

OBJECTIVE: A wide range of cardiac diseases is associated with inflammation. "Inflamed" heart tissue is infiltrated with pro-inflammatory macrophages which extensively secrete matrix metalloproteinase 9 (MMP9), a regulator of extracellular matrix turnover. As MMP9 is released from macrophages in a latent form, it requires activation. The present study addresses the role of cardiomyocytes in the course of this activation process. METHODS AND RESULTS: In mono- and co-cultures of pro-inflammatory rat macrophages (bone marrow-derived and peritoneal) and cardiomyocytes (H9C2 cell line) gelatin zymography demonstrated that activated macrophages robustly secreted latent pro-MMP9, whereas cardiomyocytes could not produce the enzyme. Co-culturing of the two cell species was critical for pro-MMP9 activation and was also accompanied by processing of cardiomyocyte-secreted pro-MMP2. A cascade of pro-MMP9 activation was initiated on macrophage membrane with pro-MMP2 cleavage. Namely, pro-inflammatory macrophages expressed an active membrane type 1 MMP (MT1MMP), which activated pro-MMP2, which in turn converted pro-MMP9. Downregulation of MT1MMP in macrophages by siRNA abolished activation of both pro-MMP2 and pro-MMP9 in co-culture. In addition, both cell species secreted MMP13 as a further pro-MMP9 activator. In co-culture, activation of pro-MMP13 occurred on membranes of macrophages and was enhanced in presence of active MMP2. Using incubations with recombinant MMPs and isolated macrophage membranes, we demonstrated that while both MMP2 and MMP13 individually had the ability to activate pro-MMP9, their combined action provided a synergistic effect. CONCLUSION: Activation of pro-MMP9 in a co-culture of pro-inflammatory macrophages and cardiomyocytes was the result of a complex interaction of several MMPs on the cell membrane and in the extracellular space. Both cell types contributed critically to pro-MMP9 processing.

Fatty acid desaturase 2 determines the lipidomic landscape and steroidogenic function of the adrenal gland.

Corticosteroids regulate vital processes, including stress responses, systemic metabolism, and blood pressure. Here, we show that corticosteroid synthesis is related to the polyunsaturated fatty acid (PUFA) content of mitochondrial phospholipids in adrenocortical cells. Inhibition of the rate-limiting enzyme of PUFA synthesis, fatty acid desaturase 2 (FADS2), leads to perturbations in the mitochondrial lipidome and diminishes steroidogenesis. Consistently, the adrenocortical mitochondria of Fads2-/- mice fed a diet with low PUFA concentration are structurally impaired and corticoid levels are decreased. On the contrary, FADS2 expression is elevated in the adrenal cortex of obese mice, and plasma corticosterone is increased, which can be counteracted by dietary supplementation with the FADS2 inhibitor SC-26192 or icosapent ethyl, an eicosapentaenoic acid ethyl ester. In humans, FADS2 expression is elevated in aldosterone-producing adenomas compared to non-active adenomas or nontumorous adrenocortical tissue and correlates with expression of steroidogenic genes. Our data demonstrate that FADS2-mediated PUFA synthesis determines adrenocortical steroidogenesis in health and disease.

Associations of Tissue and Soluble LOX-1 with Human Abdominal Aortic Aneurysm.

Background Indication for prophylactic surgical abdominal aortic aneurysm (AAA) repair depends on the maximal aortic diameter. The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is the major receptor for uptake of oxidized low-density lipoprotein cholesterol and is implicated in atherosclerosis. A soluble form of LOX-1 (sLOX-1) has been discussed as a novel biomarker in coronary artery disease and stroke. Herein, we assessed the regulation of aortic LOX-1 as well as the diagnostic and risk stratification potential of sLOX-1 in patients with AAA. Methods and Results Serum sLOX-1 was assessed in a case-control study in AAA (n=104) and peripheral artery disease (n=104). sLOX-1 was not statistically different between AAA and peripheral artery disease but was higher in AAA (ß=1.28, P=0.04) after adjusting for age, atherosclerosis, type 2 diabetes, prescription of statins, ß-blockers, ACE inhibitors, and therapeutic anticoagulation. sLOX-1 was not associated with the aortic diameter, AAA volume, or the thickness of the intraluminal thrombus. Aortic LOX-1 mRNA expression tended to be higher in AAA when compared with disease, and expression was positively associated with cleaved caspase-3, smooth muscle actin, collagen, and macrophage content. Conclusions In AAA, sLOX-1 was differently affected by age, cardiometabolic diseases, and corresponding medical therapies. Comparison with nonatherosclerotic disease would be beneficial to further elucidate the diagnostic potential of sLOX-1, although it was not useful for risk stratification. Aneurysmal LOX-1 mRNA expression was increased and positively associated with smooth muscle cells and collagen content, suggesting that LOX-1 is eventually not deleterious in human AAA and could counteract AAA rupture.

Aprotinin does not Impair Vascular Function in Patients Undergoing Coronary Artery Bypass Graft Surgery.

Bleeding is a major complication in coronary artery bypass graft surgery. Antifibrinolytic agents like serine protease inhibitor aprotinin can decrease postoperative bleeding and complications of cardiac surgery. However, the effects of aprotinin on vascular function are not completely elucidated. We compared the ex vivo vascular function of left internal mammary arteries from patients undergoing coronary artery bypass graft surgery with and without intraoperative application of aprotinin using a Mulvany Myograph. Human internal mammary arteries were treated with aprotinin ex vivo and tested for changes in vascular function. We analyzed the impact of aprotinin on vascular function in rat aortic rings. Finally, impact of aprotinin on expression and activity of endothelial nitric oxide synthase was tested in human endothelial cells. Intraoperative application of aprotinin did not impair ex vivo vascular function of internal mammary arteries of patients undergoing coronary artery bypass graft surgery. Endothelium-dependent and -independent relaxations were not different in patients with or without aprotinin after nitric oxide synthase blockade. A maximum vasorelaxation of 94.5%±11.4vs. 96.1%±5.5% indicated a similar vascular smooth muscle function in both patient groups (n=13 each). Long-term application of aprotinin under physiological condition preserved vascular function of the rat aorta. In vitro application of increasing concentrations of aprotinin on human endothelial cells resulted in a similar expression and activity of endothelial nitric oxide synthase. In conclusion, intraoperative and ex vivo application of aprotinin does not impair the endothelial function in human internal mammary arteries and experimental models.

Targeting inflammation in hypertension.

PURPOSE OF REVIEW: Hypertension remains a global health and socioeconomic burden. Immune mechanisms are now recognized as integral part of the multifactorial etiology of hypertension and related organ damage. The present review addresses inflammatory pathways and immune targets in hypertension, which may be important for an immunomodulatory treatment of hypertension aside from lowering arterial pressure. RECENT FINDINGS: Anti-inflammatory interventions targeting single interleukins or almost the entire immune system show different beneficial effects. While immunomodulation (targeting specific portion of immune system) shows beneficial outcomes in certain groups of hypertensives, this does not pertain to immunosuppression (targeting entire immune system). Immunomodulatory interventions improve outcomes of hypertension independent of arterial pressure. The studies reveal interleukins, such as interleukin (IL)-1ß and IL-17 as targets of immunomodulation. Besides interleukins, targeting αvß-3 integrin and matrix metalloproteinase-2 or using experimental cell-therapy demonstrate beneficial effects in hypertensive organ damage. The NLR family pyrin domain containing 3 (NLRP3) inflammasome/IL-1ß/endothelial cell/T-cell axis seems to be an important mediator in sustained inflammation during hypertension. SUMMARY: Although immunomodulation may be advantageous as a causal therapy in hypertension, targeting immune networks rather than single interleukins appears of major importance. Further research is required to better identify these networks and their links to human hypertension.

Targeting cardiomyocyte ADAM10 ectodomain shedding promotes survival early after myocardial infarction.

After myocardial infarction the innate immune response is pivotal in clearing of tissue debris as well as scar formation, but exaggerated cytokine and chemokine secretion with subsequent leukocyte infiltration also leads to further tissue damage. Here, we address the value of targeting a previously unknown a disintegrin and metalloprotease 10 (ADAM10)/CX3CL1 axis in the regulation of neutrophil recruitment early after MI. We show that myocardial ADAM10 is distinctly upregulated in myocardial biopsies from patients with ischemia-driven cardiomyopathy. Intriguingly, upon MI in mice, pharmacological ADAM10 inhibition as well as genetic cardiomycyte-specific ADAM10 deletion improves survival with markedly enhanced heart function and reduced scar size. Mechanistically, abolished ADAM10-mediated CX3CL1 ectodomain shedding leads to diminished IL-1ß-dependent inflammation, reduced neutrophil bone marrow egress as well as myocardial tissue infiltration. Thus, our data shows a conceptual insight into how acute MI induces chemotactic signaling via ectodomain shedding in cardiomyocytes.

NOX4 mRNA correlates with plaque stability in patients with carotid artery stenosis.

Carotid artery stenosis (CAS) develops from atherosclerotic lesions and plaques. Plaque rupture or stenosis may result in occlusion of the carotid artery. Accordingly, the asymptomatic disease becomes symptomatic, characterized by ischemic stroke or transient ischemic attacks, indicating an urgent need for better understanding of the underlying molecular mechanisms and eventually prevent symptomatic CAS. NOX4, a member of the NADPH oxidase family, has anti-atherosclerotic and anti-inflammatory properties in animal models of early atherosclerosis. We hypothesized that NOX4 mRNA expression is linked to protective mechanisms in CAS patients with advanced atherosclerotic lesions as well. Indeed, NOX4 mRNA expression is lower in patients with symptomatic CAS. A low NOX4 mRNA expression is associated with an increased risk of the development of clinical symptoms. In fact, NOX4 appears to be linked to plaque stability, apoptosis and plaque hemorrhage. This is supported by cleaved caspase-3 and glycophorin C and correlates inversely with plaque NOX4 mRNA expression. Even healing of a ruptured plaque appears to be connected to NOX4, as NOX4 mRNA expression correlates to fibrous cap collagen and is reciprocally related to MMP9 activity. In conclusion, low intra-plaque NOX4 mRNA expression is associated with an increased risk for symptomatic outcome and with reduced plaque stabilizing mechanisms suggesting protective effects of NOX4 in human advanced atherosclerosis.

Developmental endothelial locus-1 protects from hypertension-induced cardiovascular remodeling via immunomodulation.

The causative role of inflammation in hypertension-related cardiovascular diseases is evident and calls for development of specific immunomodulatory therapies. We tested the therapeutic efficacy and mechanisms of action of developmental endothelial locus-1 (DEL-1), an endogenous antiinflammatory factor, in angiotensin II- (ANGII-) and deoxycorticosterone acetate-salt-induced (DOCA-salt-induced) cardiovascular organ damage and hypertension. By using mice with endothelial overexpression of DEL-1 (EC-Del1 mice) and performing preventive and interventional studies by injecting recombinant DEL-1 in mice, we showed that DEL-1 improved endothelial function and abrogated aortic adventitial fibrosis, medial thickening, and loss of elastin. DEL-1 also protected the mice from cardiac concentric hypertrophy and interstitial and perivascular coronary fibrosis and improved left ventricular function and myocardial coronary perfusion. DEL-1 prevented aortic stiffness and abolished the progression of hypertension. Mechanistically, DEL-1 acted by inhibiting αvß3 integrin-dependent activation of pro-MMP2 in mice and in human isolated aorta. Moreover, DEL-1 stabilized αvß3 integrin-dependent CD25+FoxP3+ Treg numbers and IL-10 levels, which were associated with decreased recruitment of inflammatory cells and reduced production of proinflammatory cytokines in cardiovascular organs. The demonstrated effects and immune-modulating mechanisms of DEL-1 in abrogation of cardiovascular remodeling and progression of hypertension identify DEL-1 as a potential therapeutic factor.

Induction of Heme Oxygenase-1 Is Linked to the Severity of Disease in Human Abdominal Aortic Aneurysm.

Background Rupture of abdominal aortic aneurysm (rAAA) is associated with high case fatality rates, and risk of rupture increases with the AAA diameter. Heme oxygenase-1 (gene HMOX1, protein HO-1) is a stress-induced protein and induction has protective effects in the vessel wall. HMOX1-/- mice are more susceptible to angiotensin II-induced AAA formation, but the regulation in human nonruptured and ruptured AAA is only poorly understood. Our hypothesis proposed that HO-1 is reduced in AAA and lowering is inversely associated with the AAA diameter. Methods and Results AAA walls from patients undergoing elective open repair (eAAA) or surgery because of rupture (rAAA) were analyzed for aortic HMOX1/HO-1 expression by quantitative real-time polymerase chain reaction and Western blot. Aortas from patients with aortic occlusive disease served as controls. HMOX1/HO-1 expression was 1.1- to 7.6-fold upregulated in eAAA and rAAA. HO-1 expression was 3-fold higher in eAAA specimen with a diameter >84.4 mm, whereas HO-1 was not different in rAAA. Other variables that are known for associations with AAA and HO-1 induction were tested. In eAAA, HO-1 expression was negatively correlated with aortic collagen content and oxidative stress parameters H2O2 release, oxidized proteins, and thiobarbituric acid reactive substances. Serum HO-1 concentrations were analyzed in patients with eAAA, and maximum values were found in an aortic diameter of 55 to 70 mm with no further increase >70 mm, compared with <55 mm. Conclusions Aortic HO-1 expression was increased in eAAA and rAAA. HO-1 increased with the severity of disease but was additionally connected to less oxidative stress and vasoprotective mechanisms.

Overexpression of dimethylarginine dimethylaminohydrolase 1 protects from angiotensin II-induced cardiac hypertrophy and vascular remodeling.

Cardiovascular complications are the leading cause of death, and elevated levels of asymmetric dimethyarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, are implicated in their pathophysiology. We investigated the role of dimethylarginine dimethylaminohydrolase 1 (DDAH1), an enzyme hydrolyzing ADMA, in prevention of cardiovascular remodeling during hypertension. We hypothesized that the animals overexpressing DDAH1 will be protected from angiotensin II (ANG II)-induced end organ damage. Angiotensin II (ANG II) was infused in two doses: 0.75 and 1.5 mg/kg/day in DDAH1 transgenic mice (DDAH1 TG) and wild-type (WT) littermates for 2 or 4 wk. Echocardiography was performed in the first and fourth weeks of the infusion, systolic blood pressure (SBP) was measured weekly, and cardiac hypertrophy and vascular remodeling was assessed by histology. Increase in SBP after 1 wk of ANG II infusion was not different between the groups, whereas TG mice had lower SBP at later time points. TG mice were protected from cardiovascular remodeling after 2 wk of ANG II infusion in the high dose and after 4 wk in the moderate dose. TG mice had higher left ventricular lumen-to-wall ratio, lower cardiomyocyte cross-sectional area, and less interstitial fibrosis compared with WT controls. In aorta, TG mice had less adventitial fibrosis, lower medial thickness with preserved elastin content, lower counts of inflammatory cells, lower levels of active matrix metalloproteinase-2, and showed better endothelium-dependent relaxation. We demonstrated that overexpression of DDAH1 protects from ANG II-induced cardiovascular remodeling and progression of hypertension by preserving endothelial function and limiting inflammation.NEW & NOTEWORTHY We showed that overexpression of dimethylarginine dimethylaminohydrolase 1 (DDAH1) protects from angiotensin II-induced cardiovascular damage, progression of hypertension, and adverse vascular remodeling in vivo. This protective effect is associated with decreased levels of asymmetric dimethylarginine, preservation of endothelial function, inhibition of cardiovascular inflammation, and lower activity of matrix metalloproteinase-2. Our findings are highly clinically relevant, because they suggest that upregulation of DDAH1 might be a promising therapeutic approach against angiotensin II-induced end organ damage.

New automatic quantification method of immunofluorescence and histochemistry in whole histological sections.

Immunofluorescent staining is a widespread tool in basic science to understand organ morphology and (patho-) physiology. The analysis of imaging data is often performed manually, limiting throughput and introducing human bias. Quantitative analysis is particularly challenging for organs with complex structure such as the kidney. In this study we present an approach for automatic quantification of fluorescent markers and histochemical stainings in whole organ sections using open source software. We validate our novel method in multiple typical challenges of basic kidney research and demonstrate its general relevance and applicability to other complex solid organs for a variety of different markers and stainings. Our newly developed software tool "AQUISTO", applied as a standard in primary data analysis, facilitates efficient large scale evaluation of cellular populations in various types of histological samples. Thereby it contributes to the characterization and understanding of (patho-) physiological processes.

Estrogen determines sex differences in adrenergic vessel tone by regulation of endothelial ß-adrenoceptor expression.

Vessels of female rats constrict less and relax more to adrenergic stimulation than vessels of males. Although we have reported that these sex-specific differences rely on endothelial ß-adrenoceptors, the role of sex hormones in ß-adrenoceptor expression and related vessel tone regulation is unknown. We investigated the role of estrogen, progesterone and testosterone on ß-adrenoceptor expression and adrenergic vessel tone regulation, along with sex-specific differences in human mammary arteries. The sex-specific differences in vasoconstriction and vasorelaxation in rat vessels were eliminated after ovariectomy in females. Ovariectomy increased vessel vasoconstriction to norepinephrine more than twofold. Vasorelaxations by isoprenaline and a ß3-agonist were reduced after ovariectomy. Estrogen, but not progesterone substitution, restored sex-specific differences in vasoconstriction and vasorelaxation. Vascular mRNA levels of ß1- and ß3- but not ß2-adrenoreceptors were higher in vessels of females compared with males. Ovariectomy reduced these differences by decreasing ß1- and ß3- but not ß2-adrenoreceptor expression in females. Consistently, estrogen substitution restored ß1- and ß3-adrenoreceptor expression. Orchiectomy or testosterone treatment affected neither vasoconstriction and vasorelaxation nor ß-adrenoceptor expression in vessels of male rats. In human mammary arteries, sex-specific differences in vasoconstriction and vasorelaxation were reduced after removal of endothelium or treatment with l-NMMA. Vessels of women showed higher levels of ß1- and ß3-adrenoceptors than in men. In conclusion, the sex-specific differences in vasoconstriction and vasorelaxation are common for rat and human vessels. In rats, these differences are estrogen but not testosterone or progesterone dependent. Estrogen determines these differences via regulation of vascular endothelial ß1- and ß3-adrenoreceptor expression. NEW & NOTEWORTHY This study proposes a mechanistic concept regulating sex-specific differences in adrenergic vasoconstriction and vasorelaxation. Estrogen increases vascular ß1- and ß3-adrenoceptor expression in female rats. This and our previous studies demonstrate that these receptors are located primarily on endothelium and when activated by norepinephrine act via nitric oxide (NO). Therefore, ß-adrenergic stimulation leads to a more pronounced vasorelaxation in females. Coactivation of endothelial ß1- and ß3-adrenoreceptors leads to higher NO release in vessels of females, ultimately blunting vasoconstriction triggered by activation of smooth muscle α-adrenoceptors.

Effects of tryptophan-containing peptides on angiotensin-converting enzyme activity and vessel tone ex vivo and in vivo.

PURPOSE: Over-activation of the renin-angiotensin axis and worsening of vascular function are critical contributors to the development of hypertension. Therefore, inhibition of angiotensin-converting enzyme (ACE), a key factor of the renin-angiotensin axis, is a first line treatment of hypertension. Besides pharmaceutical ACE inhibitors, some natural peptides have been shown to exert ACE-inhibiting properties with antihypertensive effects and potentially beneficial effects on vascular function. In this study, the ACE-inhibiting potential and effects on vascular function of tryptophan-containing peptides were evaluated. METHODS: The ACE inhibitory action and stability of tryptophan-containing peptides was tested in endothelial cells-a major source of whole body ACE activity. Furthermore, the efficacy of peptides on vascular ACE activity, as well as vessel tone was assessed both ex vivo and in vivo. RESULTS: In human umbilical vein endothelial cells (HUVEC), isoleucine-tryptophan (IW) had the highest ACE inhibitory efficacy, followed by glutamic acid-tryptophan (EW) and tryptophan-leucine (WL). Whereas none of the peptides affected basal vessel tone (rat aorta), angiotensin I-induced vasoconstriction was blocked. IW effectively inhibited aortic ACE activity ex vivo taken from SHRs after 14-weeks of oral treatment with IW. Furthermore, IW treated SHRs showed better endothelium-dependent vessel relaxation compared to placebo. CONCLUSION: This study shows strong ACE-inhibiting effects of IW, EW and WL in HUVECs and aorta. The peptides effectively counteract angiotensin-induced vasoconstriction and preserve endothelium-dependent vessel relaxation. Thus, tryptophan-containing peptides and particularly IW may serve as innovative food additives with the goal of protection from angiotensin II-induced worsening of vascular function.

Sex-difference in expression and function of beta-adrenoceptors in macrovessels: role of the endothelium.

Estrogen modulates adrenergic reactivity of macrovessels, resulting in weaker α-adrenergic vasoconstriction in females than males. However, the mechanisms governing this important sex-specific difference are not well understood. We hypothesized that vessels of females express more dilatory ß-adrenoceptors, which counteract constrictive effects of α-adrenoceptors. This hypothesis was tested using aortas of normotensive (WKY) and hypertensive rats (SHR), along with human mammary artery. Selective blockade of ß1 (CGP20712) or ß3 (SR59230A), but not ß2 (ICI118,551) adrenoceptors, greatly increased α-adrenergic constriction (norepinephrine) of aorta in female SHRs, but not in male SHRs at 12 weeks of age. Consistently, the selective ß1/ß2 (isoproterenol) and ß3-adrenergic (BRL37344) relaxation was stronger in female SHRs than in males. Removal of endothelium and use of L-NMMA abolished sex-difference in α-adrenergic constriction and ß-adrenergic relaxation. Immunostainings revealed endothelial localization of ß1- and ß3-adrenoceptors. mRNA levels of aortic ß1- and ß3-, but not ß2-adrenoceptors were markedly higher in female than in male SHRs. The sex-specific differences in α-adrenergic constriction and ß-adrenoceptor mRNA levels were age-dependent, predominantly present up to 29 weeks and disappeared at 36 weeks of age. The sex-specific difference was not strain-dependent and was similarly present in normotensive WKY rats. Human mammary artery of women showed a weaker α-adrenergic constriction than arteries of men. This sex-specific difference was prominent at 45-65 years and disappeared with aging. Our results convincingly demonstrate that female macrovessels express more dilatory ß1- and ß3-adrenoreceptors than male vessels with a predominant endothelial localization. This sex-specific difference is functionally relevant in young adults and is attenuated with aging.

Sex-specific differences in age-dependent progression of aortic dysfunction and related cardiac remodeling in spontaneously hypertensive rats.

Evidence of sex-specific differences in renin-angiotensin-system (RAS) and arterial pressure has been shown in many mammals, including spontaneously hypertensive rats (SHRs). Although SHRs have been used extensively as a leading experimental model of hypertension, the effects of sex-specific differences in RAS on aortic function and related cardiac remodeling during aging and hypertension have not been documented in detail. We examined structural and functional changes in aorta and heart of female and male SHRs at the ages of 5, 14, 29, and 36 wk. SHRs of both sexes were hypertensive from 14 wk. Aortic endothelial dysfunction and fibrosis, left ventricular (LV) hypertrophy, and cardiac fibrosis were evident at the age of 29 wk in male SHRs but first appeared only at the age of 36 wk in female SHRs. There was a pronounced delay of matrix metalloproteinase-2 activity in the aorta and heart of female SHRs, which was associated with preservation of 40% more elastin and less extensive cardiac fibrosis than in males. At 5, 29, and 36 wk of age, female SHRs showed higher levels of aortic and myocardial AT2R and MasR mRNA and decreased ANG II-mediated aortic constriction. Although female SHRs had increased relaxation to AT2R stimulation at 5 and 29 wk compared with males, this difference disappeared at 36 wk of age. This study documents sex-specific differences in the temporal progression of aortic dysfunction and LV hypertrophy in SHRs, which are independent of arterial pressure and are apparently mediated by higher AT2R expression in the heart and aorta of female SHRs.

Data of the natural and pharmaceutical angiotensin-converting enzyme inhibitor isoleucine-tryptophan as a potent blocker of matrix metalloproteinase-2 expression in rat aorta.

The present data are related to the research article entitled "Whey peptide isoleucine-tryptophan inhibits expression and activity of matrix metalloproteinase-2 in rat aorta" [1]. Here we present data on removal of endothelium from aorta, endothelium dependent aortic relaxation and inhibition of expression of pro-MMP2 by di-peptide isoleucine-tryptophan (IW). Experiments were performed in rat aortic endothelial cells (EC) and smooth muscle cells (SMC) in vitro, along with isolated rat aorta ex vivo. The cells and isolated aorta were stimulated with angiotensin II (ANGII) or angiotensin I (ANGI). ACE activity was inhibited by treatment with either IW or captopril (CA). Losartan was used as a blocker of angiotensin type-1 receptor. IW inhibited MMP2 protein expression induced with ANGI in a dose-dependent manner. IW was effective both in ECs and SMCs, as well as in isolated aorta. Similarly, captopril (CA) inhibited ANGI-induced MMP2 protein expression in both in vitro and ex vivo. Neither IW nor CA inhibited ANGII-induced MMP2 protein expression in contrast to losartan. The data also displays that removal of endothelium in isolated rat aorta abolished the endothelium-dependent relaxation induced with acetylcholine. However, SMC-dependent relaxation induced with sodium nitroprusside remained intact. Finally, the data provides histological evidence of selective removal of endothelial cells from aorta.

The Cytosolic NADPH Oxidase Subunit NoxO1 Promotes an Endothelial Stalk Cell Phenotype.

OBJECTIVE: Reactive oxygen species generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidases contribute to angiogenesis and vascular repair. NADPH oxidase organizer 1 (NoxO1) is a cytosolic protein facilitating assembly of constitutively active NADPH oxidases. We speculate that NoxO1 also contributes to basal reactive oxygen species formation in the vascular system and thus modulates angiogenesis. APPROACH AND RESULTS: A NoxO1 knockout mouse was generated, and angiogenesis was studied in cultured cells and in vivo. Angiogenesis of the developing retina and after femoral artery ligation was increased in NoxO1(-/-) when compared with wild-type animals. Spheroid outgrowth assays revealed greater angiogenic capacity of NoxO1(-/-) lung endothelial cells (LECs) and a more tip-cell-like phenotype than wild-type LECs. Usually signaling by the Notch pathway switches endothelial cells from a tip into a stalk cell phenotype. NoxO1(-/-) LECs exhibited attenuated Notch signaling as a consequence of an attenuated release of the Notch intracellular domain on ligand stimulation. This release is mediated by proteolytic cleavage involving the α-secretase ADAM17. For maximal activity, ADAM17 has to be oxidized, and overexpression of NoxO1 promoted this mode of activation. Moreover, the activity of ADAM17 was reduced in NoxO1(-/-) LECs when compared with wild-type LECs. CONCLUSIONS: NoxO1 stimulates α-secretase activity probably through reactive oxygen species-mediated oxidation. Deletion of NoxO1 attenuates Notch signaling and thereby promotes a tip-cell phenotype that results in increased angiogenesis.

Whey peptide Isoleucine-Tryptophan inhibits expression and activity of matrix metalloproteinase-2 in rat aorta.

Aortic stiffness is an independent risk factor for development of cardiovascular diseases. Activation of renin-angiotensin-aldosterone system (RAAS) including angiotensin converting enzyme (ACE) activity leads to overproduction of angiotensin II (ANGII) from its precursor angiotensin I (ANGI). ANGII leads to overexpression and activation of matrix metalloproteinase-2 (MMP2), which is critically associated with pathophysiology of aortic stiffness. We previously reported that the whey peptide Isoleucine-Tryptophan (IW) acts as a potent ACE inhibitor. Herein, we critically elucidate the mechanism of action by which IW causes inhibition of expression and activity of MMP2 in aortic tissue. Effects of IW on expression and activity of MMP2 were assessed on endothelial and smooth muscle cells (ECs and SMCs) in vitro and ex vivo (isolated rat aorta). As controls we used the pharmaceutical ACE inhibitor - captopril and the ANGII type 1 receptor blocker - losartan. In vitro, both ANGII and ANGI stimulation significantly (P<0.01) increased expression of MMP2 assessed with western blot. Similarly, to captopril IW significantly (P<0.05) inhibited ANGI, but not ANGII mediated increase in expression of MMP2, while losartan also blocked effects of ANGII. Signaling pathways regulating MMP2 expression in ECs and SMCs were similarly inhibited after treatment with IW or captopril. In ECs IW significantly (P<0.05) inhibited JNK pathway, whereas in SMCs JAK2/STAT3 pathway, assessed with western blot. In vitro findings were fully consistent with results in isolated rat aorta ex vivo. Moreover, IW not only inhibited the MMP2 expression, but also its activation assessed with gelatin zymography. Our findings demonstrate that IW effectively inhibits expression and activation of MMP2 in rat aorta by decreasing local conversion of ANGI to ANGII. Thus, similar to pharmaceutical ACE inhibitor captopril the dipeptide IW may effectively inhibit ACE activity and prevent the age and hypertension associated rise of aortic stiffness.

A novel role of endothelium in activation of latent pro-membrane type 1 MMP and pro-MMP-2 in rat aorta.

AIMS: Aortic stiffness is an independent risk factor for progression of cardiovascular diseases. Degradation of elastic fibres in aorta due to angiotensin II (ANGII)-stimulated overactivation of latent membrane type 1 matrix metalloproteinase (MT1MMP) and matrix metalloproteinase-2 (MMP2) is regarded to represent an important cause of aortic stiffness. Therefore, clarification of the causal mechanisms triggering the overactivation of these MMPs is of utmost importance. This study addresses the endothelium as a novel key activator of latent pro-MT1MMP and pro-MMP2 in rat aorta. METHODS AND RESULTS: Using a co-culture model of rat aortic endothelial cells (ECs) and smooth muscle cells (SMCs), we found that ANGII stimulation resulted in activation of latent pro-MT1MMP and pro-MMP2 in SMCs exclusively when co-cultured with ECs (assessed with western blot and gelatin zymography, respectively). EC-specific AT1 receptor stimulation triggered endothelin-1 release and paracrine action on SMCs. Endothelin-1 increased expression and activity of pro-protein convertase furin in SMCs via endothelin receptor type A (assessed with qPCR and furin activity assay, respectively). Consequently, furin acted in two ways. First, it increased the activation of latent pro-MT1MMP and, second, it activated pro-αvß3 integrin. Both pathways led to overactivation of latent pro-MMP2. In vitro findings in the co-culture model were fully consistent with the ex vivo findings obtained in isolated rat aorta. CONCLUSIONS: We propose that the endothelium under ANGII stimulation acts as a novel and key activator of latent pro-MT1MMP and pro-MMP2 in SMCs of rat aorta. Therefore, endothelium may critically contribute to pathophysiology of aortic stiffness.