Expression of vascular endothelial growth factor C in human pterygium

Vascular endothelial growth factor C (VEGF-C) and its receptor VEGFR-3 mediate lymphangiogenesis. In this study, we analyzed the expression of VEGF-C and VEGFR-3 as well as lymphatic vessels in the pterygium and normal conjunctiva of humans. Fifteen primary nasal pterygia and three normal bulbar conjunctivas, surgically removed, were examined in this study. The lymphatic vessel density (LVD) and blood vessel density were determined by the immunolabeling of D2-40 and CD31, markers for lymphatic and blood vessels, respectively. VEGF-C and VEGFR-3 expression in pterygial and conjunctival tissue proteins was detected by Western blotting and were evaluated using immunohistochemistry. The LVD was significantly higher in the pterygium than normal conjunctiva (p < 0.05). Western blot demonstrated high-level expression of VEGF-C and VEGFR-3 in the pterygium compared with normal conjunctiva. VEGF-C immunoreactivity was detected in the cytoplasm of pterygial and normal conjunctival epithelial cells. The number of VEGF-C-immunopositive cells in pterygial epithelial cells was significantly higher than in normal conjunctival cells (p < 0.05). VEGFR-3 immunoreactivity was localized in the D2-40-positive lymphatic endothelial cells. The present findings suggest the potential role of VEGF-C in the pathogenesis and development of a pterygium through lymphangiogenesis and the VEGF-C/VEGFR-3 pathway as a novel therapeutic target for the human pterygium.     

Functional Local Renin-Angiotensin System in Human and Rat Periodontal Tissue

The initiation or progression of periodontitis might involve a local renin-angiotensin system (RAS) in periodontal tissue. The aim of this study was to further characterize the local RAS in human and rat periodontal tissues between healthy and periodontally-affected tissue. Components of the RAS were investigated using in vitro, ex vivo and in vivo experiments involving both human and Wistar rat periodontium. Although not upregulated when challenged with P. gingivalis-lipopolysaccharide, human gingival and periodontal ligament fibroblasts expressed RAS components. Likewise, healthy and inflamed human gingiva expressed RAS components, some of which were shown to be functional, yet no differences in expression were found between healthy and diseased gingiva. However, in inflamed tissue the immunoreactivity was greater for the AT₁R compared to AT₂R in fibroblasts. When compared to healthy tissue, ACE activity was increased in human gingiva from volunteers with gingivitis. Human-gingiva homogenates generated Ang II, Ang 1-9 and Ang 1-7 when incubated with precursors. In gingiva homogenates, Ang II formation from Ang I was nearly abolished only when captopril and chymostatin were combined. Ang 1-7 formation was significantly greater when human gingiva homogenates were incubated with chymostatin alone compared to incubation without any inhibitor, only captopril, or captopril and chymostatin. In rat gingiva, RAS components were also found; their expression was not different between healthy and experimentally induced periodontitis (EP) groups. However, renin inhibition (aliskiren) and an AT₁R antagonist (losartan) significantly blocked EP-alveolar-bone loss in rats. Collectively, these data are consistent with the hypothesis that a local RAS system is not only present but is also functional in both human and rat periodontal tissue. Furthermore, blocking AT₁R and renin can significantly prevent periodontal bone loss induced by EP in rats.     

The predominant role of brain angiotensinogen and angiotensin in environmentally induced hypertension

Rats exposed chronically to a cold environment (5 degrees C/4 degrees F) develop hypertension. This cold-induced hypertension (CIH) is a non-genetic, non-pharmacological, non-surgical model of environmentally induced hypertension in rats. The renin-angiotensin system (RAS) appears to play a role in both initiating and/or maintaining the high blood pressure in CIH. The goal of the present study was to evaluate the role of central and peripheral circulating RAS components, angiotensinogen (AGT), angiotensin-converting enzyme (ACE) and angiotensin (Ang) II, in CIH. Seventy-two Sprague-Dawley adult male rats were used. Thirty-six rats were kept in cold room at 5 degrees C while the other 36 were at 24 degrees C as controls for 5 weeks. Systolic blood pressure (SBP) was recorded by tail cuff. The SBP was increased in rats exposed to cold within 1 week, and this increase was significant for the next 2-5 weeks of the cold exposure (p<0.01). Three subgroups of the cold-treated and control rats (n=12) were sacrificed at 1, 3 and 5 weeks. The brain and liver were removed and plasma was saved. The AGT mRNA significantly increased in the hypothalamus and liver in cold-treated rats from the first week of exposure to cold, and was maintained throughout the time of exposure to cold (n=4, p<0.01). The AGT protein levels in the brain, liver and plasma did not differ significantly between cold-treated and control rats (p>0.05, n=4). The hypothalamic Ang II levels were significantly increased, whereas plasma Ang II levels significantly decreased, in the rats of 5 weeks of cold exposure (n=8, p<0.05). Plasma ACE significantly increased in the rats of 1 week of cold exposure (p<0.05, n=12). The results show differential regulation of RAS components, AGT, ACE and Ang II, between brain and periphery in cold-exposed rats. We conclude that the exposure to low temperature initially increases plasma RAS but with continuous exposure to cold, the brain RAS maintains the hypertension, probably by sustained sympathetic activation, which would provide increased metabolism but also vasoconstriction leading to hypertension.     

Localization of the renin-angiotensin system in the bovine ovary: cyclic variation of the angiotensin II receptor expression.

Angiotensin (Ang) II may modulate reproductive function in the bovine ovary. Therefore, expression and localization of a local ovarian renin-angiotensin system (RAS) were investigated by elucidating the influence of the estrus cycle, pregnancy, and the presence of follicular cysts. Receptor analysis and autoradiography were used to characterize and localize Ang II receptors. Cyclic variations in the density of ovarian Ang II receptors were found with a higher value in estrus than in diestrus. The density in ovaries with follicular cysts was in the same order of magnitude as in estrus. The Ang II receptor type 2 (AT(2)) dominated in all three groups. Autoradiography showed that the majority of antral follicles and follicular cysts had intense AT(2) receptor binding in the theca externa. Binding was less intense in the theca interna, whereas there was no binding in the granulosa layer. In the corpora lutea, the AT(2) receptor was dominant in the capsule and in connective tissue infoldings, whereas no binding was observed in the luteal tissue. The type 1 Ang II receptor (AT(1)) was dominant in the stroma and showed no cyclic changes. Angiotensin-converting enzyme (ACE) activity was detected in all aspirated follicular fluids and homogenates of ovarian tissue. Autoradiography showed that most of the ACE was localized on endothelial cells. Renin immunoreactivity was found in granulosa and thecal cells of antral follicles and in luteal cells. Furthermore, solitary cells in the stroma, presumably macrophages, displayed intense staining. Our finding of cyclic changes support the concept of an active and regulated RAS in the bovine ovary.     

核质双向转运受体Importin13 在翼状胬肉中的表达及意义

目的:探讨核质双向转运蛋白Importin13(IPO13)在翼状胬肉中的表达及意义。方法:收集2015年1月至2015年6月在中南大学湘雅医院眼科门诊确诊为"翼状胬肉"并行手术切除患者标本5例作为翼状胬肉组,收集人正常球结膜组织5例作为人正常结膜组织组,采用免疫组织化学方法检测两组结膜上皮和翼状胬肉组织IPO13、核转录因子p63、ABCG2、CD133的表达。结果:IPO13及p63表达定位于结膜上皮和翼状胬肉组织上皮基底细胞核内,ABCG2表达定位于结膜上皮和翼状胬肉上皮基底细胞层细胞浆及细胞膜,CD133表达定位于结膜上皮和翼状胬肉上皮基底细胞层细胞膜。IPO13、p63、ABCG2及CD133在人正常结膜组织中均呈低表达(光密度OD值IPO13:0.43±0.30;p63:139.09±40.15;ABCG2:63.63±22.56;CD133:70.31±33.41);在翼状胬肉中表达均明显增高(OD值IPO13:155.1±21.80;p63:617.35±87.43;ABCG2:214.03±34;CD133:201.05±46.38)。两组之间差异显著(IPO13:t'=15.87,P0.005;p63:t'=11.92,P0.005;ABCG2:t=8.15,P0.005;CD133:t=5.08,P0.005)。结论:IPO13、p63、ABCG2及CD133在翼状胬肉中高表达提示其可能在翼状胬肉异常增殖性病变中起着正向调控作用。     

Systemic hemodynamic function in humans with type 1 diabetes treated with protein kinase Cβ inhibition and renin-angiotensin system blockade: a pilot study

The protein kinase Cβ (PKCβ) system has been implicated in the deleterious vascular responses to hyperglycemia and angiotensin II (Ang?II) in experimental models of diabetes (DM). Whether these interactions are important in humans is unknown. Flow-mediated vasodilatation (FMD) was measured during clamped euglycemia and hyperglycemia, before and after randomization to PKCβ inhibition (ruboxistaurin; RBX, 32?mg daily, n = 13) or a placebo (n = 7) for 8?weeks in renin-angiotensin system (RAS) blockade-treated subjects with type 1 DM. Blood pressure responses to infused Ang?II were measured before and after randomization to RBX or a placebo. The RBX and placebo groups displayed similar clinical characteristics. Before RBX, FMD declined in response to hyperglycemia (6.8%?± 2.8% to 4.9%?± 1.8%). This effect was reversed after treatment with RBX (5.6%?± 3.1% to 6.0%?± 1.6% (within-group change, p = 0.009 (ANOVA)). No changes were observed in the placebo group. Infused Ang?II was associated with hypertensive responses in the RBX and placebo groups (p?< 0.05 (ANOVA)), and RBX did not influence this effect. In conclusion, RBX blunted the effect of hyperglycemia on FMD, suggesting that PKCβ may modulate endothelial function in type 1 DM. The lack of effect on Ang?II responses suggests that PKCβ inhibition may act through non-RAS pathways in humans with DM.     

Inflammation,oxidative stress and renin angiotensin system in atherosclerosis

Atherosclerosis is a chronic inflammatory disease associated with cardiovascular dysfunction including myocardial infarction, unstable angina, sudden cardiac death, stroke and peripheral thromboses. It has been predicted that atherosclerosis will be the primary cause of death in the world by 2020. Atherogenesis is initiated by endothelial injury due to oxidative stress associated with cardiovascular risk factors including diabetes mellitus, hypertension, cigarette smoking, dyslipidemia, obesity, and metabolic syndrome. The impairment of the endothelium associated with cardiovascular risk factors creates an imbalance between vasodilating and vasoconstricting factors, in particular, an increase in angiotensin Ⅱ(Ang Ⅱ) and a decrease in nitric oxide. The renin-angiotensin system(RAS), and its primary mediator Ang Ⅱ, also have a direct influence on the progression of the atherosclerotic process via effects on endothelial function, inflammation, fibrinolytic balance, and plaque stability. Anti-inflammatory agents [statins, secretory phospholipase A2 inhibitor, lipoprotein-associated phospholipase A2 inhibitor, 5-lipoxygenase activating protein, chemokine motif ligand-2, C-C chemokine motif receptor 2 pathway inhibitors, methotrexate, IL-1 pathway inhibitor and RAS inhibitors(angiotensin-converting enzyme inhibitors)], Ang Ⅱ receptor blockers and ranin inhibitors may slow inflammatory processes and disease progression. Several studies in human using anti-inflammatory agents and RAS inhibitors revealed vascular benefits and reduced progression of coronary atherosclerosis in patients with stable angina pectoris; decreased vascular inflammatory markers, improved common carotid intima-media thickness and plaque volume in patients with diagnosed atherosclerosis. Recent preclinical studies have demonstrated therapeutic efficacy of vitamin D analogs paricalcitol in Apo E-deficient atherosclerotic mice.     

Angiotensin II induces NF-kappa B activation in HUVEC via the p38MAPK pathway

Angiotensin II (Ang II) is the main active peptide of the renin–angiotensin system (RAS), producing a number of inflammatory mediators that lead to endothelial dysfunction and the progression of atherosclerosis. Ang II-induced NF-κB nuclear translocation plays a pivotal role in this response. This study examines the NF-κB activation mechanism elicited by Ang II in human umbilical vein endothelial cells (HUVEC). Electrophoretic mobility shift assays and Western blotting revealed that Ang II, signaling via AT₁, produces a time-dependent increase in NF-κB DNA binding and IκB degradation. These results also demonstrate that Ang II leads to MAPK phosphorylation and p38MAPK pathway-induced NF-κB activation. Furthermore, AT₁ is required for p38MAPK phosphorylation induced by Ang II. This study provides evidence that Ang II elicits NF-κB activation via the p38MAPK pathway in HUVEC.     

New insights and perspectives on intrarenal renin-angiotensin system: focus on intracrine/intracellular angiotensin II

Although renin, the rate-limiting enzyme of the renin-angiotensin system (RAS), was first discovered by Robert Tigerstedt and Bergman more than a century ago, the research on the RAS still remains stronger than ever. The RAS, once considered to be an endocrine system, is now widely recognized as dual (circulating and local/tissue) or multiple hormonal systems (endocrine, paracrine and intracrine). In addition to the classical renin/angiotensin I-converting enzyme (ACE)/angiotensin II (Ang II)/Ang II receptor (AT₁/AT₂) axis, the prorenin/(Pro)renin receptor (PRR)/MAP kinase axis, the ACE2/Ang (1-7)/Mas receptor axis, and the Ang IV/AT₄/insulin-regulated aminopeptidase (IRAP) axis have recently been discovered. Furthermore, the roles of the evolving RAS have been extended far beyond blood pressure control, aldosterone synthesis, and body fluid and electrolyte homeostasis. Indeed, novel actions and underlying signaling mechanisms for each member of the RAS in physiology and diseases are continuously uncovered. However, many challenges still remain in the RAS research field despite of more than one century's research effort. It is expected that the research on the expanded RAS will continue to play a prominent role in cardiovascular, renal and hypertension research. The purpose of this article is to review the progress recently being made in the RAS research, with special emphasis on the local RAS in the kidney and the newly discovered prorenin/PRR/MAP kinase axis, the ACE2/Ang (1-7)/Mas receptor axis, the Ang IV/AT₄/IRAP axis, and intracrine/intracellular Ang II. The improved knowledge of the expanded RAS will help us better understand how the classical renin/ACE/Ang II/AT₁ receptor axis, extracellular and/or intracellular origin, interacts with other novel RAS axes to regulate blood pressure and cardiovascular and kidney function in both physiological and diseased states.     

A new look at the renin-angiotensin system--focusing on the vascular system

The renin–angiotensin system (RAS), critically involved in the control of blood pressure and volume homeostasis, is a dual system comprising a circulating component and a local tissue component. The rate limiting enzyme is renin, which in the circulating RAS derives from the kidney to generate Ang II, which in turn regulates cardiovascular function by binding to AT₁ and AT₂ receptors on cardiac, renal and vascular cells. The tissue RAS can operate independently of the circulating RAS and may be activated even when the circulating RAS is suppressed or normal. A functional tissue RAS has been identified in brain, kidney, heart, adipose tissue, hematopoietic tissue, gastrointestinal tract, liver, endocrine system and blood vessels. Whereas angiotensinsinogen, angiotensin converting enzyme (ACE), Ang I and Ang II are synthesized within these tissues, there is still controversy as to whether renin is produced locally or whether it is taken up from the circulation, possibly by the (pro)renin receptor. This is particularly true in the vascular wall, where expression of renin is very low. The exact function of the vascular RAS remains elusive, but may contribute to fine-tuning of vascular tone and arterial structure and may amplify vascular effects of the circulating RAS, particularly in pathological conditions, such as in hypertension, atherosclerosis and diabetes. New concepts relating to the vascular RAS have recently been elucidated including: (1) the presence of functionally active Ang-(1-7)-Mas axis in the vascular system, (2) the importance of the RAS in perivascular adipose tissue and cross talk with vessels, and (3) the contribution to vascular RAS of Ang II derived from immune and inflammatory cells within the vascular wall. The present review highlights recent progress in the RAS field, focusing on the tissue system and particularly on the vascular RAS.     

The pregnancy-induced increase of plasma angiotensin-(1-7) is blunted in gestational diabetes

BACKGROUND AND OBJECTIVE: It has been shown that the circulating Renin-Angiotensin System (RAS) is activated during normal pregnancy, but little is known about RAS in pregnancies complicated by gestational diabetes (GDM). GDM is considered not merely a temporary condition, but a harbinger of hypertension and type 2 diabetes. The aim of this study was to evaluate the circulating RAS profile in normotensive women with GDM at the third trimester of pregnancy and to compare the results with healthy pregnant and non-pregnant age-matched women. METHODS: The diagnostic criteria for GDM followed the recommendations of the American Diabetes Association. Angiotensin I (Ang I), Angiotensin II (Ang II) and Angiotensin 1-7 [Ang-(1-7)] were determined in 24 pregnant patients with GDM; 12 healthy pregnant women and 12 non-pregnant women by radioimmunoassay. RESULTS: Levels of Ang I, Ang II and Ang-(1-7) were higher in pregnant women (p<0.05), but showed a different pattern in the GDM group, in which reduced Ang-(1-7) circulating levels were found (p<0.05). This observation was confirmed by the significantly lower Ang-(1-7)/Ang I ratio (p<0.05). CONCLUSION: Our data suggest that reduced levels of the vasodilator Ang-(1-7) could be implicated in the endothelial dysfunction seen in gestational diabetic women during and after pregnancy.     

Characterization of the Cardiac Renin Angiotensin System in Oophorectomized and Estrogen-Replete mRen2.Lewis Rats

The cardioprotective effects of estrogen are well recognized, but the mechanisms remain poorly understood. Accumulating evidence suggests that the local cardiac renin-angiotensin system (RAS) is involved in the development and progression of cardiac hypertrophy, remodeling, and heart failure. Estrogen attenuates the effects of an activated circulating RAS; however, its role in regulating the cardiac RAS is unclear. Bilateral oophorectomy (OVX; n = 17) or sham-operation (Sham; n = 13) was performed in 4-week-old, female mRen2.Lewis rats. At 11 weeks of age, the rats were randomized and received either 17 β-estradiol (E2, 36 µg/pellet, 60-day release, n = 8) or vehicle (OVX-V, n = 9) for 4 weeks. The rats were sacrificed, and blood and hearts were used to determine protein and/or gene expression of circulating and tissue RAS components. E2 treatment minimized the rise in circulating angiotensin (Ang) II and aldosterone produced by loss of ovarian estrogens. Chronic E2 also attenuated OVX-associated increases in cardiac Ang II, Ang-(1–7) content, chymase gene expression, and mast cell number. Neither OVX nor OVX+E2 altered cardiac expression or activity of renin, angiotensinogen, angiotensin-converting enzyme (ACE), and Ang II type 1 receptor (AT1R). E2 treatment in OVX rats significantly decreased gene expression of MMP-9, ACE2, and Ang-(1–7) mas receptor, in comparison to sham-operated and OVX littermates. E2 treatment appears to inhibit upsurges in cardiac Ang II expression in the OVX-mRen2 rat, possibly by reducing chymase-dependent Ang II formation. Further studies are warranted to determine whether an E2-mediated reduction in cardiac chymase directly contributes to this response in OVX rats.     

Immunohistochemical study of vascular endothelial growth factor (VEGF), tumor suppressor protein (p53) and intercellular adhesion molecule (ICAM-1) in the conjunctiva of diabetic patients

Summary The expression pattern of VEGF, p53 and ICAM-1 was studied in conjunctiva of diabetic patients with and without retinopathy. All patients underwent a complete ophthalmic examination, including retinal fluorescein angiography. Indirect immunoperoxidase method was performed on 20 eyes of 20 patients with type II diabetes without DR and on 5 eyes of 5 patients with PDR. A control study was performed on 6 normal conjunctiva undertaken during cataract surgery. Immunoreactivity of VEGF, p53 and ICAM-1 was found in epithelial, fibroblast and vascular endothelial cells. For the same duration of diabetes, a strong to moderate or weak immunoreactivity was observed in the conjunctiva of patients without retinopathy. In patients with PDR, the expression was strong for all these proteins. The immunoreactivity was correlated between VEGF, p53 and ICAM-1. In the normal conjunctiva, a weak to negative immunostaining was observed. The presence of these proteins in the conjunctiva of diabetic patients without retinopathy may add new data in the pathogenesis of diabetic retinopathy. Further studies are needed to confirm this hypothesis.     

Angiotensin III: A physiological relevant peptide of the renin angiotensin system

The renin angiotensin system (RAS) is a peptide hormone system that plays an important role in the pathophysiology of various diseases, including congestive heart failure, hypertension, myocardial infarction, and diabetic nephropathy. This has led researchers to focus extensively on this system, leading to the discovery of various peptides, peptidases, receptors and signal transduction mechanisms intrinsic to the RAS. Angiotensinogen (AGT), angiotensin (Ang) II, Ang III, Ang IV, and Ang-(1–7) are the main biologically active peptides of RAS. However, most of the available studies have focused on Ang II as the likely key peptide from the RAS that directly and indirectly regulates physiological functions leading to pathological conditions. However, data from recent studies suggest that Ang III may produce physiologically relevant effects that are similar to those produced by Ang II. Hence, this review focuses on Ang III and the myriad of physiological effects that it produces in the body.     

Csk regulates angiotensin II-induced podocyte apoptosis

Increasing data have shown that angiotensin II (Ang II) perpetuates podocyte injury and promotes progression to end-stage kidney disease. The mechanism underlying Ang II-induced podocyte apoptosis has not been established. C-terminal Src kinase (Csk) is a cytoplasmic kinase that interacts with scaffolding proteins involved in cell growth, adhesion, and polarization, and the role of Csk in regulating cellular apoptosis has gradually attracted attention. This study evaluates the role of Csk in Ang II-induced podocyte apoptosis. In vivo, Wistar rats were randomly subjected to a normal saline or Ang II infusion. In vitro, we exposed differentiated mouse podocytes to Ang II. Ang II increased Csk expression and induced podocyte apoptosis, stimulated Csk translocation and binding to Caveolin-1, and stimulated decreased Fyn pY416, increased Fyn pY529, and nephrin dephosphorylation. Csk knockdown prevented Ang II-induced podocyte apoptosis, reduced Fyn kinase inactivation, and increased the interaction between nephrin and the activated form of Fyn, accompanied by a reduced interaction between Csk and Caveolin-1. These findings indicate that Ang II induces podocyte injury via a Csk-dependent pathway.     

Losartan, a specific angiotensin II receptor antagonist, increases angiotensin I and angiotensin II release from isolated rat hind legs: evidence for locally regulated renin-angiotensin system in vascular tissue.

The effect of Losartan (10(-9) to 10(-6) M) on angiotensins I and II release was examined in isolated hind legs perfused with Krebs-Ringer solution from normal and bilaterally nephrectomized rats. Losartan increased dramatically both angiotensins I (Ang I) and II (Ang II) release in a dose-dependent fashion; the maximal percent increment in Ang I and Ang II release evoked by Losartan (10(-6) M) was about +380% and +160%, respectively, in normal rat hind legs. In nephrectomized animals, Losartan elicited a marked increase in both peptides dose-dependently. There was a highly positive correlation between the released amounts of Ang I and that of Ang II altered by Losartan in either normal (r = 0.954) or nephrectomized rats (r = 0.923). These results not only confirm the existence of a functional renin-angiotensin system in vascular tissues, but also suggest that the system is regulated by locally generated Ang II.     

Modulation and function of extrarenal angiotensin receptors

Historically, physiological modulation of the activity of the renin-angiotension system (RAS) was thought to be mediated only by changes in renin secretion. Hence, altered dietary sodium (Na) intake, changes in renal perfusion pressure, and/or renal adrenoreceptor activity would lead to changes in renin release and plasma angiotensin II (Ang II) concentration, which in turn contribute to regulation of blood pressure and sodium balance. Later, it became apparent that angiotensinogen availability and Ang-converting enzyme activity are also rate-limiting factors that influence the activity of RAS. Finally, over the past few years, evidence has accumulated that indicates the number of Ang II receptors and their subtypes are of great importance in regulating the activity and function of RAS. Cloning of the Ang II receptor genes, development of specific receptor-antagonist ligands, and establishment of genetically mutated animal models have led to greater understanding of the role of Ang II receptors in the regulation of RAS function and activity. This review focuses on the functions and regulation of Ang II receptors in vascular tissues and in the adrenal gland. The authors suggest that identification of control elements for Ang II receptor expression, which are tissue-specific, may provide a basis for future therapeutic manipulation of Ang II receptors in cardiovascular disease states.