Angiotensin receptor subtype AT(1) mediates alveolar epithelial cell apoptosis in response to ANG II

Previous work from this laboratory demonstrated induction of apoptosis in lung alveolar epithelial cells (AEC) by purified angiotensin II (ANG II) and expression of mRNAs for both ANG II receptor subtypes AT(1) and AT(2) (Wang R, Zagariya A, Ibarra-Sunga O, Gidea C, Ang E, Deshmukh S, Chaudhary G, Baraboutis J, Filippatos G, and Uhal BD. Am J Physiol Lung Cell Mol Physiol 276: L885-L889, 1999.). The present study was designed to determine the ANG II receptor subtype mediating AEC apoptosis in response to ANG II. Apoptosis was induced with purified ANG II applied to the human lung AEC-derived carcinoma cell line A549 or to primary AEC isolated from Wistar rats. In both cell types, the AT(1)-selective receptor antagonists L-158809 or losartan inhibited ANG II-induced apoptosis by 90% at concentrations of 10(-8) M and 10(-7) M, respectively. The inhibition was concentration dependent with IC(50) of 10(-12) M and 10(-11) M on the primary rat AEC. In contrast, the AT(2)-selective antagonists PD-123319 or PD-126055 could not block ANG II-induced apoptosis in either cell type. In primary rat AEC, apoptosis in response to ANG II was blunted in a dose-dependent manner by the protein kinase C inhibitor chelerythrine but not by the tyrosine phosphatase inhibitor sodium orthovanadate. Together, these data indicate that AEC apoptosis in response to ANG II is mediated by receptor subtype AT(1), despite the expression of mRNAs for both AT(1) and AT(2).     

Essential role for cathepsin D in bleomycin-induced apoptosis of alveolar epithelial cells

Our earlier studies showed that bleomycin-induced apoptosis of type II alveolar epithelial cells (AECs) requires the autocrine synthesis and proteolytic processing of angiotensinogen into ANG II and that inhibitors of ANG-converting enzyme (ACEis) block bleomycin-induced apoptosis (Li X, Zhang H, Soledad-Conrad V, Zhuang J, and Uhal BD. Am J Physiol Lung Cell Mol Physiol 284: L501-L507, 2003). Given the documented role of cathepsin D (CatD) in apoptosis of other cell types, we hypothesized that CatD might be the AEC enzyme responsible for the conversion of angiotensinogen into ANG I, the substrate for ACE. Primary cultures of rat type II AECs challenged with bleomycin in vitro showed upregulation and secretion of CatD enzymatic activity and immunoreactive protein but no increases in CatD mRNA. The aspartyl protease inhibitor pepstatin A, which completely blocked CatD enzymatic activity, inhibited bleomycin-induced nuclear fragmentation by 76% and reduced bleomycin-induced caspase-3 activation by 47%. Antisense oligonucleotides against CatD mRNA reduced CatD-immunoreactive protein and inhibited bleomycin-induced nuclear fragmentation by 48%. A purified fragment of angiotensinogen (F1-14) containing the CatD and ACE cleavage sites, when applied to unchallenged AEC in vitro, yielded mature ANG II peptide and induced apoptosis. The apoptosis induced by F1-14 was inhibited 96% by pepstatin A and 77% by neutralizing antibodies specific for CatD (both P < 0.001). These data indicate a critical role for CatD in bleomycin-induced apoptosis of cultured AEC and suggest that the role(s) of CatD in AEC apoptosis include the conversion of newly synthesized angiotensinogen to ANG II.     

Abrogation of bleomycin-induced epithelial apoptosis and lung fibrosis by captopril or by a caspase inhibitor

Angiotensin-converting enzyme is involved in apoptosis of alveolar epithelial cells (Wang R, Zagariya A, Ang E, Ibarra-Sunga O, and Uhal BD. Am J Physiol Lung Cell Mol Physiol 277: L1245-L1250, 1999). This study tested the ability of the angiotensin-converting enzyme inhibitor captopril or the caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone (ZVAD-fmk) to block alveolar epithelial cell apoptosis and lung fibrosis in vivo in response to bleomycin (Bleo). Male Wistar rats received 8 U/kg of Bleo (bleomycin sulfate) or vehicle intratracheally. Subgroups of Bleo-treated rats received captopril, ZVAD-fmk, or vehicle alone. Lung collagen was assessed by picrosirius red or hydroxyproline assay at 1, 7, and 14 days post-Bleo, and apoptosis was detected by in situ end labeling (ISEL). Bleo increased alveolar septal and peribronchial collagen by 100 and 133%, respectively (both P < 0.01), by day 14 but not earlier. In contrast, ISEL was increased in alveolar and airway cells at all time points. Captopril or ZVAD-fmk inhibited collagen accumulation by 91 and 85%, respectively (P < 0. 01). Both agents also inhibited ISEL in alveoli by 99 and 81% and in airways by 67 and 63%, respectively. These data suggest that the efficacy of captopril to inhibit experimental lung fibrogenesis is related to inhibition of apoptosis. They also demonstrate the antifibrotic potential of a caspase inhibitor.     

Amiodarone induces apoptosis of human and rat alveolar epithelial cells in vitro

The antiarrhythmic amiodarone (AM) and its metabolite desethylamiodarone (Des) are known to cause AM-induced pulmonary toxicity, but the mechanisms underlying this disorder remain unclear. We hypothesized that AM might cause AM-induced pulmonary toxicity in part through the induction of apoptosis or necrosis in alveolar epithelial cells (AECs). Two models of type II pneumocytes, the human AEC-derived A549 cell line and primary AECs isolated from adult Wistar rats, were incubated with AM or Des for 20 h. Apoptotic cells were determined by morphological assessment of nuclear fragmentation with propidium iodide on ethanol-fixed cells. Necrotic cells were quantitated by loss of dye exclusion. Both AM and Des caused dose-dependent necrosis starting at 2.5 and 0.1 microg/ml, respectively, in primary rat AECs and at 10 and 5 microg/ml in subconfluent A549 cells (P < 0.05 and P < 0.01, respectively). AM and Des also induced dose-dependent apoptosis beginning at 2.5 microg/ml in the primary AECs (P < 0.05 for both compounds) and at 10 and 5 microg/ml, respectively, in the A549 cell line (P < 0.01). The two compounds also caused significant net cell loss (up to 80% over 20 h of incubation) by either cell type at drug concentrations near or below the therapeutic serum concentration for AM. The cell loss was not due to detachment but was blocked by the broad-spectrum caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone. Furthermore, the angiotensin-converting enzyme inhibitor captopril (500 ng/ml) and the angiotensin-receptor antagonist saralasin (50 microg/ml) significantly inhibited both the induction of apoptosis and net cell loss in response to AM. These results are consistent with recent work from this laboratory demonstrating potent inhibition of apoptosis in human AECs by captopril (Uhal BD, Gidea C, Bargout R, Bifero A, Ibarra-Sunga O, Papp M, Flynn K, and Filippatos G. Am J Physiol Lung Cell Mol Physiol 275: L1013-L1017, 1998). They also suggested that the accumulation of AM and/or its primary metabolite Des in lung tissue may induce cytotoxicity of AECs that might be inhibitable by angiotensin-converting enzyme inhibitors or other antagonists of the renin-angiotensin system.     

Regulation of alveolar epithelial cell survival by the ACE-2/angiotensin 1-7/Mas axis

Earlier work from this laboratory demonstrated that apoptosis of alveolar epithelial cells (AECs) requires autocrine generation of angiotensin (ANG) II. More recent studies showed that angiotensin converting enzyme-2 (ACE-2), which degrades ANGII to form ANG1-7, is protective but severely downregulated in human and experimental lung fibrosis. Here it was theorized that ACE-2 and its product ANG1-7 might therefore regulate AEC apoptosis. To evaluate this hypothesis, the AEC cell line MLE-12 and primary cultures of rat AECs were exposed to the profibrotic apoptosis inducers ANGII or bleomycin (Bleo). Markers of apoptosis (caspase-9 or -3 activation and nuclear fragmentation), steady-state ANGII and ANG1-7, and JNK phosphorylation were measured thereafter. In the absence of Bleo, inhibition of ACE-2 by small interfering RNA or by a competitive inhibitor (DX600 peptide) caused a reciprocal increase in autocrine ANGII and corresponding decrease in ANG1-7 in cell culture media (both P < 0.05) and, moreover, induced AEC apoptosis. At baseline (without inhibitor), ANG1-7 in culture media was 10-fold higher than ANGII (P < 0.01). Addition of purified ANGII or bleomycin-induced caspase activation, nuclear fragmentation, and JNK phosphorylation in cultured AECs. However, preincubation with ANG1-7 (0.1 μM) prevented JNK phosphorylation and apoptosis. Moreover, pretreatment with A779, a specific blocker of the ANG1-7 receptor mas, prevented ANG1-7 blockade of JNK phosphorylation, caspase activation, and nuclear fragmentation. These data demonstrate that ACE-2 regulates AEC survival by balancing the proapoptotic ANGII and its antiapoptotic degradation product ANG1-7. They also suggest that ANG1-7 inhibits AEC apoptosis through the ANG1-7 receptor mas.     

Bleomycin-induced apoptosis of alveolar epithelial cells requires angiotensin synthesis de novo

Primary cultures of rat type II alveolar epithelial cells (AECs) or human AEC-derived A549 cells, when exposed to bleomycin (Bleo), exhibited concentration-dependent apoptosis detected by altered nuclear morphology, fragmentation of DNA, activation of caspase-3, and net cell loss over time. In both cell culture models, exposure to Bleo caused time-dependent increases in angiotensinogen (ANGEN) mRNA. Antisense oligonucleotides against ANGEN mRNA inhibited Bleo-induced apoptosis of rat AEC or A549 cells by 83 and 84%, respectively (P < 0.01 and P < 0.05), and prevented Bleo-induced net cell loss. Apoptosis of rat AECs or A549 cells in response to Bleo was inhibited 91% by the ANG-converting enzyme inhibitor captopril or 82%, respectively, by neutralizing antibodies specific for ANG II (both P < 0.01). Antagonists of ANG receptor AT(1) (losartan, L-158809, or saralasin), but not an AT(2)-selective blocker (PD-123319), inhibited Bleo-induced apoptosis of either rat AECs (79%, P < 0.01) or A549 cells (83%, P < 0.01) and also reduced the activity of caspase-3 by 52% (P < 0.05). These data indicate that Bleo, like Fas(L) or TNF-alpha, induces transactivation of ANG synthesis de novo that is required for AEC apoptosis. They also support the theory that ANG system antagonists have potential for the blockade of AEC apoptosis in situ.     

Aldosterone induces interleukin-18 through endothelin-1, angiotensin II, Rho/Rho-kinase, and PPARs in cardiomyocytes

Aldosterone (Aldo) is recognized as an important risk factor for cardiovascular diseases. IL-18 induces myocardial hypertrophy, loss of contractility of cardiomyocytes, and apoptosis leading myocardial dysfunction. However, so far, there have been few reports concerning the interaction between Aldo and IL-18. The present study examined the effects and mechanisms of Aldo on IL-18 expression and the roles of peroxisome proliferator-activated receptor (PPAR) agonists in rat cardiomyocytes. We used cultured rat neonatal cardiomyocytes stimulated with Aldo to measure IL-18 mRNA and protein expression, Rho-kinase, and NF-kappaB activity. We also investigated the effects of PPAR agonists on these actions. Aldo, endothelin-1 (ET-1), and angiotensin II (ANG II) increased IL-18 mRNA and protein expression. Mineralocorticoid receptor antagonists, endothelin A receptor antagonist, and ANG II receptor antagonist inhibited Aldo-induced IL-18 expression. Aldo induced ET-1 and ANG II production in cultured media. Moreover, Rho/Rho-kinase inhibitor and statin inhibited Aldo-induced IL-18 expression. On the other hand, Aldo upregulated the activities of Rho-kinase and NF-kappaB. PPAR agonists attenuated the Aldo-induced IL-18 expression and NF-kappaB activity but not the Rho-kinase activity. Our findings indicate that Aldo induces IL-18 expression through a mechanism that involves, at a minimum, ET-1 and ANG II acting via the Rho/Rho-kinase and PPAR/NF-kappaB pathway. The induction of IL-18 in cardiomyocytes by Aldo, ET-1, and ANG II might, therefore, cause a deterioration of the cardiac function in an autocrine and paracrine fashion. The inhibition of the IL-18 expression by PPAR agonists might be one of the mechanisms whereby the beneficial cardiovascular effects are exerted.     

Coupling of angiotensin II AT1 receptors to neuronal NHE activity and carrier-mediated norepinephrine release in myocardial ischemia

In ischemia, cardiac sympathetic nerve endings (cSNE) release excessive amounts of norepinephrine (NE) via the nonexocytotic Na(+)-dependent NE transporter (NET). NET, normally responsible for NE reuptake into cSNE, reverses in myocardial ischemia, releasing pathological amounts of NE. This carrier-mediated NE release can be triggered by elevated intracellular Na(+) levels in the axoplasm. The fact that ischemia activates the intracellular pH regulatory Na(+)/H(+) exchanger (NHE) in cSNE is pivotal in increasing intraneuronal Na(+) and thus activating carrier-mediated NE release. Angiotensin (ANG) II levels are also significantly elevated in the ischemic heart. However, the effects of ANG II on cSNE, which express the ANG II receptor, AT(1)R, are poorly understood. We hypothesized that ANG II-induced AT(1)R activation in cSNE may be positively coupled to NHE activity and thereby facilitate the pathological release of NE associated with myocardial ischemia. We tested this hypothesis in a cSNE model, human neuroblastoma cells stably transfected with rat recombinant AT(1A) receptor (SH-SY5Y-AT(1A)). SH-SY5Y-AT(1A) constitutively expresses amiloride-sensitive NHE and the NET. NHE activity was assayed in BCECF-loaded SH-SY5Y-AT(1A) as the rate of the Na(+)-dependent alkalinization in response to an acute acidosis. ANG II activation of AT(1)R markedly increased NHE activity in SH-SY5Y-AT(1A) via a Ca(2+)-dependent pathway and promoted carrier-mediated NE release. In addition, in guinea pig cSNE expressing native AT(1)R, ANG II elicited carrier-mediated NE release. In SH-SY5Y-AT(1A) and cSNE, amiloride inhibited the ANG II-mediated release of NE. Our results provide a link between AT(1)R and NHE in cSNE, which can exacerbate carrier-mediated NE release during protracted myocardial ischemia.     

Effects of atrial natriuretic peptide, angiotensin II and III on norepinephrine uptake in the rat adrenal medulla.

The effects of atrial natriuretic peptide (ANP), angiotensin II (ANG II) and angiotensin III (ANG III) on norepinephrine (NE) uptake were studied in the adrenal medulla of the rat. One microM ANG II and 10 microM ANG III decreased NE uptake while 10 nM and 100 nM ANP increased it. Subthreshold concentrations of ANP (1 nM) blunted the inhibitory effect of 1 microM ANG II but did not modify the inhibitory effect of 10 microM ANG III. The increasing effects of 100 nM ANP on NE uptake were partially reversed by subthreshold concentrations of ANG II (1 nM) and blunted by 1 nM ANG III. The interaction between ANP and the renin-angiotensin system could contribute to modulate the sympathetic function in the adrenal medulla.     

经气管灌注血管紧张素Ⅱ导致凋亡性急性肺损伤(英文)

为研究外源性血管紧张素Ⅱ（angiotensinⅡ,ANG）在急性肺损伤和肺泡上皮细胞凋亡中的作用,经气管分别给雄性Wistar大鼠（175-200g）灌注ANG、ANG加caspase抑制剂ZVAD—fmk、ANG加ANG受体1阻断剂losartan和仅灌注磷酸盐缓冲溶液（PBS）。6或20h后在体灌洗动物肺脏,测定灌洗液中血红蛋白（hemoglobin,Hb）和荧光物质（BODIPY）标定的白蛋白含量（在灌洗前15min静脉注入BODIPY-白蛋白）。TUNEL测定显示,灌注ANG6h后,支气管和肺泡上皮细胞内标定的DNA片断显著增2H（P〈0．05）;ANG所致的DNA片断增加可被同时灌注ZVAD—fmk或losartan阻断。灌注ANG后免疫标定caspase3阳性细胞数量显著增多（P〈0．01）,ZVAD—fmk或losartan同样显著减少caspase3阳性细胞的数量。灌注ANG显著增加肺泡灌洗液中荧光标定的白蛋白（P〈0．01）和Hb的含量（P〈0．05）;ZVAD—fmk或losartan亦显著抑制荧光白蛋白和Hb含量的变化。结果表明,肺泡上皮细胞在体暴露于外源性ANG足以引起ANG受体1介导的上皮细胞凋亡和肺泡屏障损伤。     

Inward remodeling of resistance arteries requires reactive oxygen species-dependent activation of matrix metalloproteinases

Inward eutrophic remodeling is the most prevalent structural change of resistance arteries in hypertension. Sympathetic and angiotensin (ANG)-induced vasoconstriction has been associated with hypertension and with the production of matrix metalloproteinases (MMPs) and ROS. Therefore, we hypothesize that prolonged exposure to norepinephrine (NE) and ANG II induces arteriolar inward remodeling dependent on the activation of MMPs and the production of ROS. This hypothesis was tested on rat cremaster arterioles that were isolated, cannulated, pressurized, and exposed to either NE (10(-5.5) mol/l) + ANG II (10(-7) mol/l) or vehicle (control) for 4 h. The prolonged exposure to NE + ANG II induced inward remodeling, as evidenced by the reduced maximal arteriolar passive diameter observed after versus before exposure to the vasoconstrictor agonists. NE + ANG II also increased the arteriolar expression and activity of MMP-2 and the production of ROS as determined, respectively, by real-time RT-PCR, gel and in situ zymography, and the use of ROS-sensitive dyes with multiphoton microscopy. Inhibition of MMP activation (with GM-6001) or ROS production (with apocynin or tempol) prevented the NE + ANG II-induced inward remodeling. Inhibition of ROS production prevented the activation of MMPs and the remodeling process, whereas inhibition of MMP activation did not affect ROS production. These results indicate that prolonged stimulation of resistance arterioles with NE + ANG II induces a ROS-dependent activation of MMPs necessary for the development of arteriolar inward remodeling. These mechanisms may contribute to the structural narrowing of resistance vessels in hypertension.     

The design and synthesis of a potent Angiotensin II cyclic analogue confirms the ring cluster receptor conformation of the hormone Angiotensin II

The novel amide linked Angiotensin II potent cyclic analogue, c-[Sar1,Lys3,Glu5] ANG II 19 has been designed and synthesized in an attempt to test the aromatic ring clustering and the charge relay bioactive conformation we have recently suggested for ANG II. This constrained cyclic analogue was synthesized by connecting the Lys3 amino and Glu5 carboxyl side chain groups, and it was found to be potent in the rat uterus assay and in anesthetized rabbits. The central part of the molecule is fixed covalently in the conformation predicted according to the backbone bend conformational model proposed for Angiotensin II. The obtained results using a combination of 2D NMR, 1D NOE spectroscopy and molecular modeling revealed a similar Tyr4-Ile5-His6 bend, a His6-Pro7 trans configuration and a side chain aromatic ring cluster of the key aminoacids Tyr4, His6, Phe8 for c-[Sar1,Lys3,Glu5] ANG II as it has been found for ANG II (Matsoukas, J. H.; Hondrelis, J.; Keramida, M.; Mavromoustakos, T.; Markriyannis, A.; Yamdagni, R.; Wu, Q.; Moore, G. J. J. Biol. Chem. 1994, 269, 5303). Previous study of the conformational properties of the Angiotensin II type I antagonist [Hser(gamma-OMe)8] ANG II (Matsoukas, J. M.; Agelis, G.; Wahhab, A.; Hondrelis, J.; Panagiotopoulos. D.; Yamdagni, R.; Wu, Q.; Mavromoustakos, T.; Maia, H.; Ganter, R.; Moore, G. J. J. Med. Chem. 1995, 38, 4660) using 1-D NOE spectroscopy coupled with the present study of the same type of lead antagonist Sarilesin revealed that the Tyr4-Ile5-His6 bend, a conformational property found in Angiotensin II is not present in type I antagonists. The obtained results provide an important conformational difference between Angiotensin II agonists and type I antagonists. It appears that our synthetic attempt to further support our proposed model was successful and points out that the charge relay system and aromatic ring cluster are essential stereoelectronic features for Angiotensin II to exert its biological activity.     

Defining the differential sensitivity to norepinephrine and angiotensin II in the ovine uterine vasculature

The intact ovine uterine vascular bed (UVB) is sensitive to α-agonists and refractory to angiotensin II (ANG II) during pregnancy; the converse occurs in the systemic circulation. The mechanism(s) responsible for these differences in uterine sensitivity are unclear and may reflect predominance of nonconstricting AT(2) receptors (AT(2)R) in uterine vascular smooth muscle (UVSM). The contribution of the placental vasculature also is unclear. Third generation and precaruncular/placental arteries from nonpregnant (n = 16) and term pregnant (n = 23) sheep were used to study contraction responses to KCl, norepinephrine (NE), and ANG II (with/without ATR specific inhibitors) and determine UVSM ATR subtype expression and contractile protein content. KCl and NE increased third generation and precaruncular/placental UVSM contractions in a dose- and pregnancy-dependent manner (P ≤ 0.001). ANG II only elicited modest contractions in third generation pregnant UVSM (P = 0.04) and none in precaruncular/placental UVSM. Moreover, compared with KCl and NE, ANG II contractions were diminished ≥ 5-fold. Whereas KCl and ANG II contracted third generation>precaruncular/placental UVSM, NE-induced contractions were similar throughout the UVB. However, each agonist increased third generation contractions ≥ 2-fold at term, paralleling increased actin/myosin and cellular protein content (P ≤ 0.01). UVSM AT(1)R and AT(2)R expression was similar throughout the UVB and unchanged during pregnancy (P > 0.1). AT(1)R inhibition blocked ANG II-mediated contractions; AT(2)R blockade, however, did not enhance contractions. AT(2)R predominate throughout the UVB of nonpregnant and pregnant sheep, contributing to an inherent refractoriness to ANG II. In contrast, NE elicits enhanced contractility throughout the ovine UVB that exceeds ANG II and increases further at term pregnancy.     

Acid-base regulation and control of ventilation in human pregnancy

The purposes of this review were twofold: to apply modern physicochemical principles to explain changes in acid-base regulation and the control of ventilation in human pregnancy; and to demonstrate the value of pregnancy as a model for the study of endocrine effects on physiological control systems. Application of P.A. Stewart's approach (P.A. Stewart. Can. J. Physiol. Pharmacol. 61: 1444-1461, 1983) shows that lower values of plasma hydrogen ion concentration ([H+]) observed at rest and in association with exercise in pregnancy are the result of lower values for carbon dioxide tension (Pco2) and total weak acid ([A(tot)]). This effect is partly offset by a lower strong ion difference ([SID]). The ability to predict plasma [H+] at rest and following strenuous exercise in pregnancy (J.G. Kemp, F.A. Greer, and L.A. Wolfe. J. Appl. Physiol. 83: 644-651, 1997) supports the validity of Stewart's approach. Jennings and associates (D.B. Jennings. Can. J. Physiol. Pharmacol. 72: 1499-1512, 1994) have further demonstrated in animal models the involvement of plasma osmolality and circulating levels of angiotensin II (ANG II) and arginine vasopressin (AVP) in the chemical control of ventilation. We hypothesize that pregnancy-induced increases in respiratory sensitivity to carbon dioxide are the combined result of reduced plasma osmolality, reduced cerebrospinal fluid [SID], and augmented circulating levels of progesterone, ANG II, and AVP.     

ANG II potentiates mitogenic effect of norepinephrine in vascular muscle cells: role of FGF-2

We examined the possible cooperation between norepinephrine (NE) and ANG II on proliferation of cultured vascular smooth muscle cells (VSMCs) and the involved cellular mechanisms. Nanomolar NE concentrations stimulated VSMC proliferation through a prazosin-sensitive effect. The pretreatment of cells with 100 nM ANG II for 24 h significantly potentiated the NE-induced VSMC proliferation; this potentiating effect of ANG II was blocked by losartan but was unaffected by the AT(2) receptor antagonist PD-123177. ANG II pretreatment also potentiated the increase in inositol phosphate turnover and upregulated the cell expression of fibroblast growth factor (FGF-2) induced by NE. Anti-FGF-2 neutralizing antibodies prevented the potentiating effect of ANG II on NE-induced cell growth. Both ANG II and NE stimulated extracellular signal-related kinase (ERK1) activation, but an ANG II potentiation of the effect of NE on ERK1 activity was not detectable. Moreover, ANG II significantly increased protein synthesis but did not potentiate the hypertrophic effect of NE. These findings demonstrate that ANG II and NE cooperate in promoting VSMC growth and that FGF-2 upregulation is involved in this effect.     

Autophagy: The missing link between non-enzymatically glycated proteins inducing apoptosis and premature senescence of endothelial cells?

In a series of studies into the fate of endothelial cells exposed to non-enzymatically glycated collagen I, a model of cytotoxic molecules relevant to diabetic vasculopathy, we demonstrate that cells either undergo apoptosis or become prematurely senescent despite relatively spared telomeres and telomerase activity. Our most recent work shows that long-lived advanced glycation end product (AGE)-modified proteins induce 1) lysosomal permeabilization leading to the inefficiency of autophagy due to the reduced digestion (early) and non-fusion (later) of lysosomes with phagosomes—a frustrated autophagy; and 2) accumulation of lipid mediators, such as ceramide and sphingosine-1-phosphate, known to be involved in autophagic cell death. Under the experimental conditions described here, the seesaw relations between premature senescence and apoptosis are integrated by autophagy, which plays a novel function of a cellular switch between states of premature senescence and apoptosis.

Addendum to: Patschan SA, Chen J, Polotskaia A, Mendelev N, Cheng J, Patschan D, Goligorsky MS. Lipid mediators of autophagy in stress-induced premature senescence of endothelial cells. Am J Physiol Heart Circ Physiol 2008; In press.

and

Patschan S, Chen J, Gealekman O, Krupincza K, Wang M, Shu L, Shayman JA, Goligorsky MS. Mapping mechanisms and charting the time course of premature cell senescence and apoptosis: lysosomal dysfunction and ganglioside accumulation in endothelial cells. Am J Physiol Renal Physiol 2008; 294:F100-9.     

Fetal uninephrectomy in male sheep alters the systemic and renal responses to angiotensin II infusion and AT1R blockade

Fetal uninephrectomy (uni-x) at 100 days of gestation results in compensatory nephrogenesis in the remaining kidney, resulting in a 30% reduction in total nephron number in male sheep. Recently, we showed that uni-x males at 6 mo of age have elevated arterial pressure, reduced renal blood flow (RBF), glomerular filtration rate (GFR), and low plasma renin levels (Singh R, Denton K, Bertram J, Jefferies A, Head G, Lombardo P, Schneider-Kolsky M, Moritz K. J Hypertens 27: 386-396, 2009; Singh R, Denton K, Jefferies A, Bertram J, Moritz K. Clin Sci (Lond) 118: 669-680, 2010). We hypothesized this was due to upregulation of the intrarenal renin-angiotensin system (RAS). In this study, renal responses to ANG II infusion and ANG II type 1 receptor (AT1R) blockade were examined in the same 6-mo-old male sheep. Uni-x animals had reduced levels of renal tissue and plasma renin and ANG II. Renal gene expression of renin, and gene and protein levels of AT1R and AT2R, were significantly lower in uni-x animals. In response to graded ANG II infusion, sham animals had the expected decrease in conscious RBF and GFR. Interestingly, the response was biphasic in uni-x sheep, with GFR initially decreasing, but then increasing at higher ANG II doses (34 ± 7%; P(group × treatment) < 0.001), due to a paradoxical decrease in renal vascular resistance (P(group × treatment) < 0.001). In response to AT1R blockade, while GFR and RBF responded similarly between groups, there was a marked increase in sodium excretion in uni-x compared with sham sheep (209 ± 35 vs. 25 ± 12%; P < 0.001). In conclusion, in 6-mo-old male sheep born with a single kidney, these studies demonstrate that this is a low-renin form of hypertension, in which responses to ANG II are perturbed and the intrarenal RAS is downregulated.     

ANG II induces apoptosis of human vascular smooth muscle via extrinsic pathway involving inhibition of Akt phosphorylation and increased FasL expression

In addition to well-documented vascular growth-promoting effects, ANG II exerts proapoptotic effects that are poorly understood. IGF-1 is a potent survival factor for human vascular smooth muscle cells (hVSMC), and its antiapoptotic effects are mediated via the IGF-1 receptor (IGF-1R) through a signaling pathway involving phosphatidylinositol 3-kinase and Akt. We hypothesized that there would be cross talk between ANG II proapoptotic effects and IGF-1 survival effects in hVSMC. To investigate ANG II-induced apoptosis and the potential involvement of IGF-1, we exposed quiescent and nonquiescent hVSMC to ANG II. ANG II induced apoptosis only in nonquiescent cells but stimulated hypertrophy in quiescent cells. ANG II-induced apoptosis was characterized by marked inhibition of Akt phosphorylation and stimulation of membrane Fas ligand (FasL) expression, caspase-8 activation, and a reduction in soluble FasL expression. Adenovirally mediated overexpression of Akt rescued hVSMC from ANG II-induced apoptosis. IGF-1R activation increased Akt phosphorylation and soluble FasL expression, and these effects were completely blocked by coincubating hVSMC with ANG II. In conclusion, ANG II-induced apoptosis of hVSMC is characterized by marked inhibition of Akt phosphorylation and stimulation of an extrinsic cell death signaling pathway via upregulation of membrane FasL expression, caspase-8 activation, and a reduction in soluble FasL expression. Furthermore, ANG II antagonizes the antiapoptotic effect of IGF-1 by blocking its ability to increase Akt phosphorylation and soluble FasL. These findings provide novel insights into ANG II-induced apoptotic signaling and have significant implication for understanding ANG II-induced remodeling in hypertension and atherosclerosis.     

Effect of ANG II on endothelial cell apoptosis and survival and its impact on skeletal muscle angiogenesis after electrical stimulation

We have previously shown that skeletal muscle angiogenesis induced by electrical stimulation is significantly attenuated when SS-13BN/Mcwi rats are fed a high-salt diet. This effect was associated with a large increase in endothelial cell (EC) apoptosis. We hypothesized that the low levels of ANG II during high-salt diet would increase EC apoptosis and consequently diminish the angiogenic response. To test this hypothesis, a series of in vitro and in vivo studies was performed. EC apoptosis and viability were evaluated after incubation with ANG II under serum-free conditions. After 24 h of incubation, ANG II increased EC viability and Bcl-2-to-Bax ratio along with a dose-dependent decrease in EC apoptosis. This effect was blocked by the ANG II type 1 receptor antagonist losartan. To confirm our in vitro results, ANG II (3 ng.kg(-1).min(-1)) was chronically infused in rats fed a high-salt diet (4% NaCl). ANG II decreased EC apoptosis and produced a significant increase (40%) in skeletal muscle angiogenesis after electrical stimulation. These in vivo results were in agreement with our in vitro results and demonstrate that the attenuation of ANG II levels during a high-salt diet may induce EC apoptosis and consequently block the angiogenic response induced by electrical stimulation. Furthermore, under normal conditions, ANG II increases EC viability and protects EC from apoptosis possibly by inactivation of the mitochondrial apoptotic pathway.     

Whole body norepinephrine kinetics in ANG II-salt hypertension in the rat

The purpose of this study was to investigate total body norepinephrine (NE) kinetics as an index of global sympathetic nervous system (SNS) outflow in a rat model of chronic ANG II-salt hypertension. Male Sprague-Dawley rats fed a 0.4% (normal salt, NS) or 2% (HS) NaCl diet were instrumented with arterial and venous catheters. After 5 days of recovery and a 3-day control period, ANG II (150 ng.kg(-1).min(-1)) was given subcutaneously by minipump for 14 days. Plasma NE levels and total body NE spillover and clearance were determined on control day 3 and ANG II infusion days 7 and 14 using radioisotope dilution principles. To perform this analysis, 3H-NE and NE were measured in arterial plasma after a 90-min infusion of tracer amounts of 3H-NE. Mean arterial pressure (MAP) was similar during the control period in NS and HS rats; however, MAP increased to a higher level in HS rats. During the control period, plasma NE tended to be lower in rats on HS, whereas NE clearance tended to be higher in HS rats. As a result NE spillover was similar in NS and HS rats during the control period. In NS rats, plasma NE, NE spillover, and NE clearance were unchanged by ANG II. In contrast, in rats on the HS diet, plasma NE and NE spillover increased during ANG II infusion, whereas NE clearance was unchanged. In conclusion, a HS diet alone or ANG II infusion in animals fed NS do not affect global sympathetic outflow. However, the additional hypertensive response to ANG II in animals fed HS is accompanied by SNS activation.