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Zhiying Shan,
Jasenka Zubcevic,
Peng Shi,
Joo Y Jun,
Ying Dong,
Tatiane M Murça,
Gwyneth J Lamont,
Adolfo Cuadra,
Wei Yuan,
Yanfei Qi, Qiuhong Li,
Julian F R Paton,
Michael J Katovich,
Colin Sumners,
Mohan K Raizada
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ABSTRACT: AT1 receptor subtype a (AT1Ra) expression is increased in the nucleus of the solitary tract (NTS) in spontaneously hypertensive rat (SHR) compared with Wistar Kyoto controls. However, the chronic role of AT1Ra in the NTS for cardiovascular control is not well understood. In this study, we investigated the hypothesis that the NTS AT1Ra is involved in the neural regulation of the peripheral inflammatory status and linked with hypertension. Transduction of brain neuronal cultures with recombinant adeno-associated virus type 2 (AAV2)-AT1R-small hairpin RNA (shRNA) resulted in a 72% decrease in AT1Ra mRNA and attenuated angiotensin II-induced increase in extracellular signal-regulated kinase 1/2 phosphorylation and neuronal firing. Specific NTS microinjection of AAV2-AT1R-shRNA vector in the SHR resulted in a ≈30 mm Hg increase in the mean arterial pressure compared with control vector-injected animals (Sc-shRNA: 154±4 mm Hg; AT1R-shRNA: 183±10 mm Hg) and induced a resetting of the baroreflex control of heart rate to higher mean arterial pressure. In addition, AAV2-AT1R-shRNA-treated SHRs exhibited a 74% decrease in circulating endothelial progenitor cells (CD90(+), CD4(-)/CD5(-)/CD8(-)) and a 300% increase in the circulating inflammatory cells, including CD4(+)(+)CD8(+), CD45(+)/3(+) T lymphocytes, and macrophages (CD68(+)). As a result, the endothelial progenitor cell/inflammatory cells ratio was decreased by 8- to 15-fold in the AT1R-shRNA-treated SHR. However, identical injection of AAV2-AT1R-shRNA into the NTS of Wistar Kyoto rats had no effect on mean arterial pressure and inflammatory cells. These observations suggest that increased expression of the AT1Ra in SHR NTS may present a counterhypertensive mechanism involving inflammatory/angiogenic cells.
Hypertension 04/2013; · 6.21 Impact Factor
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Jasenka Zubcevic,
Joo Y Jun,
Gwyneth Lamont,
Tatiane M Murça,
Peng Shi,
Wei Yuan,
Fan Lin,
Jessica Marulanda Carvajal, Qiuhong Li,
Colin Sumners,
Mohan K Raizada,
Zhiying Shan
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ABSTRACT: The importance of the (pro)renin receptor (PRR) in the function of the central nervous system is increasingly evident because PRR seems to play a role in neuronal control of cardiovascular function. PRR expression is elevated in the nucleus of the solitary tract (NTS) of spontaneously hypertensive rats (SHR). In this study, we tested the hypothesis that altered activity of PRR in the NTS is linked to hypertension. Eight weeks of chronic knockdown of the NTS PRR, using recombinant adeno-associated virus type 2 (AAV2)-PRR-small hairpain RNA (shRNA)-mediated gene transduction, caused a significant increase in mean arterial pressure (MAP) in the SHR (shRNA, 173±5; Control, 151±6 mm Hg) but not in Wistar Kyoto rats (shRNA, 108±7; Control, 106±6 mm Hg). The MAP elevation in the SHR was associated with decreased inflammatory markers tumor necrosis factor-α, interleukin-6, C-C motif ligand 5, and their transcription factor, nuclear factor-κB. Consistent with the pressor effects of the PRR knockdown, acute bilateral NTS injection of human renin (2 pmol/side) decreased MAP and heart rate (HR) in SHR (ΔMAP, -38±4 mm Hg; Δheart rate, -40±10 bpm), with negligible responses in Wistar Kyoto rats (ΔMAP, -4±3 mm Hg; Δheart rate, -12±7 bpm). These effects in SHR were attenuated (80%) by prorenin handle region peptide but were not affected by angiotensin II type 1 or angiotensin II type 2 receptor blockers. Finally, PRR activation in SHR neuronal cultures by prorenin activated nuclear factor-κB and increased mRNA levels of interleukin-1β (250-fold), tumor necrosis factor-α (32-fold), interleukin-6 (35-fold), C-C motif ligand 5 (12-fold), and interleukin-10 (7-fold) in a nuclear factor-κB-dependent but angiotensin II type 1 receptor-independent manner. Therefore, NTS PRR mediates antihypertensive effects via an angiotensin II-independent mechanism in SHR, which involves stimulation of the nuclear factor-κB-cytokine signaling pathway.
Hypertension 01/2013; · 6.21 Impact Factor
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ABSTRACT: Gene therapy has a distinct potential to treat kidney diseases. However, the efficient transduction of a significant number of renal cells by viral vectors has been difficult to accomplish. Previous studies have indicated that adeno-associated virus (AAV) can transduce renal cells with variable and suboptimal efficiency. Since new and innovative mutants of AAV are now available, we compared their efficacy in transducing rat kidneys. We compared five types of AAV mutants (AAV2 muttriple, AAV2 sextuple, AAV8 mut447, AAV8 mut733, and AAV9 mut446) that carried a green fluorescence protein (GFP) reporter gene. A pressure microinjection technique was used to inject either 1.5x10(11) vector genome (vg) of AAV mutants or three dose of AAV2 sextuple into the renal cortex. The microinjection approach has not been utilized in AAV-mediated renal gene transfer thus far. Slow and sustained microinjection enables continuous administration of the viral vector to the kidney cortex and limits any damage to the kidney, as the tip of a glass micropipette is very small. Three weeks after injection, the kidneys were collected and evaluated for GFP expression. Among the various mutated AAV serotypes studied, only AAV2 sextuple showed a robust GFP expression in renal tissue. The AAV2 sextuple serotype appears to be an efficient gene transfer vector to preferentially target the renal tubular epithelial cells. A combination of the AAV2 sextuple and the microinjection technique holds the key to the future of therapeutic treatments for kidney diseases. © 2012 The Authors Clinical and Experimental Pharmacology and Physiology © 2012 Wiley Publishing Asia Pty Ltd.
Clinical and Experimental Pharmacology and Physiology 12/2012; · 1.85 Impact Factor
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Wen-Tao Deng,
Astra Dinculescu, Qiuhong Li,
Sanford L Boye,
Jie Li,
Marina S Gorbatyuk,
Jijing Pang,
Vince A Chiodo,
Michael T Matthes,
Douglas Yasumura,
Li Liu,
Fowzan S Alkuraya,
Kang Zhang,
Douglas Vollrath,
Matthew M LaVail,
William W Hauswirth
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ABSTRACT: The absence of Mertk in RCS rats results in defective RPE phagocytosis, accumulation of outer segment (OS) debris in the subretinal space, and subsequent death of photoreceptors. Previous research utilizing Mertk gene replacement therapy in RCS rats provided proof of concept for treatment of this form of recessive retinitis pigmentosa (RP); however, the beneficial effects on retinal function were transient. In the present study, we evaluated whether delivery of a MERTK transgene using a tyrosine-mutant AAV8 capsid could lead to more robust and longer-term therapeutic outcomes than previously reported.
An AAV8 Y733F vector expressing a human MERTK cDNA driven by a RPE-selective promoter was administrated subretinally at postnatal day 2. Functional and morphological analyses were performed at 4 months and 8 months post-treatment. Retinal vasculature and Müller cell activation were analyzed by quantifying acellular capillaries and glial fibrillary acidic protein immunostaining, respectively.
Electroretinographic responses from treated eyes were more than one-third of wild-type levels and OS were well preserved in the injection area even at 8 months. Rescue of RPE phagocytosis, prevention of retinal vasculature degeneration, and inhibition of Müller cell activation were demonstrated in the treated eyes for at least 8 months.
This research describes a longer and much more robust functional and morphological rescue than previous studies. We also demonstrate for the first time that an AAV8 mutant capsid serotype vector has a substantial therapeutic potential for RPE-specific gene delivery. These results suggest that tyrosine-mutant AAV8 vectors hold promise for the treatment of individuals with MERTK-associated RP.
Investigative ophthalmology & visual science 03/2012; 53(4):1895-904. · 3.43 Impact Factor
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Astra Dinculescu,
Jackie Estreicher,
Juan C Zenteno,
Tomas S Aleman,
Sharon B Schwartz,
Wei Chieh Huang,
Alejandro J Roman,
Alexander Sumaroka, Qiuhong Li,
Wen-Tao Deng, [......],
Vince A Chiodo,
Andy Neeley,
Xuan Liu,
Xinhua Shu,
Margarita Matias-Florentino,
Beatriz Buentello-Volante,
Sanford L Boye,
Artur V Cideciyan,
William W Hauswirth,
Samuel G Jacobson
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ABSTRACT: Autosomal recessive retinitis pigmentosa (RP), a heterogeneous group of degenerations of the retina, can be due to mutations in the MFRP (membrane-type frizzled-related protein) gene. A patient with RP with MFRP mutations, one of which is novel and the first splice site mutation reported, was characterized by noninvasive retinal and visual studies. The phenotype, albeit complex, suggested that this retinal degeneration may be a candidate for gene-based therapy. Proof-of-concept studies were performed in the rd6 Mfrp mutant mouse model. The fast-acting tyrosine-capsid mutant AAV8 (Y733F) vector containing the small chicken β-actin promoter driving the wild-type mouse Mfrp gene was used. Subretinal vector delivery on postnatal day 14 prevented retinal degeneration. Treatment rescued rod and cone photoreceptors, as assessed by electroretinography and retinal histology at 2 months of age. This AAV-mediated gene delivery also resulted in robust MFRP expression predominantly in its normal location within the retinal pigment epithelium apical membrane and its microvilli. The clinical features of MFRP-RP and our preliminary data indicating a response to gene therapy in the rd6 mouse suggest that this form of RP is a potential target for gene-based therapy.
Human gene therapy 12/2011; 23(4):367-76. · 4.20 Impact Factor
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ABSTRACT: We hypothesized that moderate cardiac-selective overexpression of the angiotensin II type 2 receptor (AT2R) would protect the myocardium from ischaemic injury after a myocardial infarction (MI) induced by coronary artery ligation. For in vitro studies, adenoviral vector expressing genomic DNA of AT2R and enhanced green fluorescence protein (EGFP) was used to overexpress AT2R in rat neonatal cardiac myocytes. Expression of AT2R, measured by real-time PCR and immunostaining, demonstrated efficient transduction of AT2R in a dose-dependent pattern. The AT2R constitutively induced apoptosis in rat neonatal cardiac myocytes in dose-dependent patterns. For in vivo studies, 4 × 10(10) vector genomes (vg) of recombinant adeno-associated virus serotype 9 (rAAV9)-chicken β actin promoter-AT2R was injected into the left ventricle of 5-day-old Sprague-Dawley rats. At 6 weeks of age, hearts were harvested and expression of AT2R determined by real-time PCR and Western blotting. Expression was increased onefold over control hearts, and no apoptosis was detected. Two subsequent in vivo studies were performed. In a prevention study, 4 × 10(10) vg of rAAV9-CBA-AT2R was injected into the left ventricle of 5-day-old Sprague-Dawley rats and MI was induced at 6 weeks of age. For a post-treatment study, 4 × 10(10) vg of rAAV9-CBA-AT2R was administrated to the peri-infarcted myocardium area immediately after MI in 6-week-old animals. For both in vivo studies, cardiac functions were assessed using echocardiography and haemodynamic measurements 4 weeks after coronary artery ligation. In the in vivo studies, the rats subjected to MI showed significant decreases in fractional shortening and rate of change of left ventricular pressure, with increased left ventricular end-diastolic pressure and ventricular hypertrophy. For the prevention study, the moderate cardiac-selective overexpression of AT2R attenuated these MI-induced impairments and also caused a decrease in ventricular wall thinning. In the post-treatment study, the overexpression of AT2R partly reversed the MI-induced cardiac dysfunction. Myocardial infarction also induced the upregulation of angiotensin II type 1 receptor, angiotensin-converting enzyme and collagen I mRNA expression, all of which were attenuated by the overexpression of AT2R. It is concluded that moderate cardiac-selective overexpression of AT2R protects heart function from ischaemic injury, which may be mediated, at least in part, through modulation of components of the cardiac renin-angiotensin system and collagen levels in the myocardium.
Experimental physiology 10/2011; 97(1):89-101. · 3.17 Impact Factor
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Amrisha Verma,
Zhiying Shan,
Bo Lei,
Lihui Yuan,
Xuan Liu,
Takahiko Nakagawa,
Maria B Grant,
Alfred S Lewin,
William W Hauswirth,
Mohan K Raizada, Qiuhong Li
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ABSTRACT: Despite evidence that hyperactivity of the vasodeleterious axis (ACE/angiotensin II (Ang II)/AT1 receptor) of the renin-angiotensin system (RAS) is associated with the pathogenesis of diabetic retinopathy (DR) use of the inhibitors of this axis has met with limited success in the control of this pathophysiology. We investigated the hypothesis that enhancing the local activity of the recently established protective axis of the RAS, ACE2/Ang-(1-7), using adeno-associated virus (AAV)-mediated gene delivery of ACE2 or Ang-(1-7) would confer protection against diabetes-induced retinopathy. Genes expressing ACE2 and Ang-(1-7) were cloned in AAV vector. The effects of ocular AAV-ACE2/Ang-(1-7) gene transfer on DR in diabetic eNOS(-/-) mice and Sprague-Dawley (SD) rats were examined. Diabetes was associated with approximately tenfold and greater than threefold increases in the ratios of ACE/ACE2 and AT1R/Mas mRNA levels in the retina respectively. Intraocular administration of AAV-ACE2/Ang-(1-7) resulted in significant reduction in diabetes-induced retinal vascular leakage, acellular capillaries, infiltrating inflammatory cells and oxidative damage in both diabetic mice and rats. Our results demonstrate that DR is associated with impaired balance of retinal RAS. Increased expression of ACE2/Ang-(1-7) overcomes this imbalance and confers protection against DR. Thus, strategies enhancing the protective ACE2/Ang-(1-7) axis of RAS in the eye could serve as a novel therapeutic target for DR.
Molecular Therapy 07/2011; 20(1):28-36. · 6.87 Impact Factor
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Waichi Sato,
Katsuyuki Tanabe,
Tomoki Kosugi,
Kelly Hudkins,
Miguel A Lanaspa,
Li Zhang,
Martha Campbell-Thompson, Qiuhong Li,
David A Long,
Charles E Alpers,
Takahiko Nakagawa
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ABSTRACT: Vascular endothelial growth factor A (VEGF-A) can play both beneficial and deleterious roles in renal diseases, where its specific function might be determined by nitric oxide bioavailability. The complexity of VEGF-A in renal disease could in part be accounted for by the distinct roles of its two receptors; VEGFR1 is involved in the inflammatory responses, whereas VEGFR2 predominantly mediates angiogenesis. Because nondiabetic chronic renal disease is associated with capillary loss, we hypothesized that selective stimulation of VEGFR2 could be beneficial in this setting. However, VEGFR2 activation may be deleterious in the presence of nitric oxide deficiency. We systematically overexpressed a mutant form of VEGF-A binding only VEGFR2 (Flk-sel) using an adeno-associated virus-1 vector in wild-type and eNOS knockout mice and then induced renal injury by uninephrectomy. Flk-sel treatment increased angiogenesis and lowered blood pressure in both mouse types. Flk-sel overexpression caused mesangial injury with increased proliferation associated with elevated expression of PDGF, PDGF-β receptor, and VEGFR2; this effect was greater in eNOS knockout than in wild-type mice. Flk-sel also induced tubulointerstitial injury, with some tubular epithelial cells expressing α-smooth muscle actin, indicating a phenotypic evolution toward myofibroblasts. In conclusion, prestimulation of VEGFR2 can potentiate subsequent renal injury in mice, an effect enhanced in the setting of nitric oxide deficiency.
American Journal Of Pathology 07/2011; 179(1):155-66. · 4.89 Impact Factor
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ABSTRACT: The vasodegenerative phase of diabetic retinopathy is likely caused by endothelial dysfunction and reduced endothelial repair. Migration of endothelial progenitor cells (EPCs) into areas of vascular injury is critical to vascular repair. This key function, often defective in diabetes, is largely mediated by nitric oxide (NO), which is known to be inactivated by superoxide produced by NADPH oxidase. The authors tested the hypothesis that either increasing eNOS expression or inhibiting NADPH oxidase would restore the reparative function in diabetic EPCs.
Peripheral blood was obtained from healthy (n = 27) and diabetic (n = 31) persons, and CD34(+) cells were isolated. Expression and activation of eNOS and NADPH oxidase and intracellular levels of NO, superoxide, and peroxynitrite were evaluated. cGMP production and migration to SDF-1α were also determined. Reparative function was evaluated in a mouse model of retinal ischemia-reperfusion injury.
Diabetic EPCs demonstrate reduced eNOS expression and decreased NO bioavailability and migration in response to SDF-1α. Increasing eNOS expression in diabetic cells by AVE3085 resulted in increased peroxynitrite levels and, therefore, did not enhance NO-mediated functions in vitro and in vivo. Expression of Nox2, NADPH oxidase activity, and superoxide levels were higher in diabetic than in nondiabetic EPCs. Pretreatment with apocynin or gp91ds-tat increased NO bioavailability without increasing eNOS activity in response to SDF-1α. Ex vivo NADPH oxidase inhibition in diabetic cells restored migratory function in vitro and enhanced their homing to ischemic retinal vasculature in vivo.
The NADPH oxidase system is a promising target for correcting vasoreparative dysfunction in diabetic EPCs.
Investigative ophthalmology & visual science 06/2011; 52(8):5093-104. · 3.43 Impact Factor
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Marc C Peden,
Jeff Min,
Craig Meyers,
Zachary Lukowski, Qiuhong Li,
Sanford L Boye,
Monica Levine,
William W Hauswirth,
Ramakrishna Ratnakaram,
William Dawson,
Wesley C Smith,
Mark B Sherwood
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ABSTRACT: The current method of delivering gene replacement to the posterior segment of the eye involves a three-port pars plana vitrectomy followed by injection of the agent through a 37-gauge cannula, which is potentially wrought with retinal complications. In this paper we investigate the safety and efficacy of delivering adeno-associated viral (AAV) vector to the suprachoroidal space using an ab externo approach that utilizes an illuminated microcatheter.
6 New Zealand White rabbits and 2 Dutch Belted rabbits were used to evaluate the ab externo delivery method. sc-AAV5-smCBA-hGFP vector was delivered into the suprachoroidal space using an illuminated iTrackTM 250A microcatheter. Six weeks after surgery, the rabbits were sacrificed and their eyes evaluated for AAV transfection using immunofluorescent antibody staining of GFP.
Immunostaining of sectioned and whole-mounted eyes demonstrated robust transfection in all treated eyes, with no fluorescence in untreated control eyes. Transfection occurred diffusely and involved both the choroid and the retina. No apparent adverse effects caused by either the viral vector or the procedure itself could be seen either clinically or histologically.
The ab externo method of delivery using a microcatheter was successful in safely and effectively delivering a gene therapy agent to the suprachoroidal space. This method presents a less invasive alternative to the current method of virally vectored gene delivery.
PLoS ONE 01/2011; 6(2):e17140. · 4.09 Impact Factor
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Hilda Petrs-Silva,
Astra Dinculescu, Qiuhong Li,
Wen-Tao Deng,
Ji-Jing Pang,
Seok-Hong Min,
Vince Chiodo,
Andy W Neeley,
Lakshmanan Govindasamy,
Antonette Bennett,
Mavis Agbandje-McKenna,
Li Zhong,
Baozheng Li,
Giridhara R Jayandharan,
Arun Srivastava,
Alfred S Lewin,
William W Hauswirth
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ABSTRACT: Vectors based on adeno-associated virus serotype 2 (AAV2) have been used extensively in many gene-delivery applications, including several successful clinical trials for one type of Leber congenital amaurosis in the retina. Many studies have focused on improving AAV2 transduction efficiency and cellular specificity by genetically engineering its capsid. We have previously shown that vectors-containing single-point mutations of capsid surface tyrosines in serotypes AAV2, AAV8, and AAV9 displayed significantly increased transduction efficiency in the retina compared with their wild-type counterparts. In the present study, we evaluated the transduction characteristics of AAV2 vectors containing combinations of multiple tyrosine to phenylalanine mutations in seven highly conserved surface-exposed capsid tyrosine residues following subretinal or intravitreal delivery in adult mice. The multiply mutated vectors exhibited different in vivo transduction properties, with some having a unique ability of transgene expression in all retinal layers. Such novel vectors may be useful in developing valuable new therapeutic strategies for the treatment of many genetic diseases.
Molecular Therapy 11/2010; 19(2):293-301. · 6.87 Impact Factor
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ABSTRACT: The mechanisms that drive the development of diabetic nephropathy remain undetermined. Only 30-40% of patients with diabetes mellitus develop overt nephropathy, which suggests that other contributing factors besides the diabetic state are required for the progression of diabetic nephropathy. Endothelial dysfunction is associated with human diabetic nephropathy and retinopathy, and advanced diabetic glomerulopathy often exhibits thrombotic microangiopathy, including glomerular capillary microaneurysms and mesangiolysis, which are typical manifestations of endothelial dysfunction in the glomerulus. Likewise, diabetic mice with severe endothelial dysfunction owing to deficiency of endothelial nitric oxide synthase develop progressive nephropathy and retinopathy similar to the advanced lesions observed in humans with diabetes mellitus. Additionally, inhibitors of the renin-angiotensin system fail to be renoprotective in some individuals with diabetic nephropathy (due in part to aldosterone breakthrough) and in some mouse models of the disease. In this Review, we discuss the clinical and experimental evidence that supports a role for endothelial nitric oxide deficiency and subsequent endothelial dysfunction in the progression of diabetic nephropathy and retinopathy. If endothelial dysfunction is the key factor required for diabetic nephropathy, then agents that improve endothelial function or raise intraglomerular nitric oxide level could be beneficial in the treatment of diabetic nephropathy.
Nature Reviews Nephrology 11/2010; 7(1):36-44. · 7.09 Impact Factor
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Zhiying Shan,
Peng Shi,
Adolfo E Cuadra,
Ying Dong,
Gwyneth J Lamont, Qiuhong Li,
Dale M Seth,
L Gabriel Navar,
Michael J Katovich,
Colin Sumners,
Mohan K Raizada
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ABSTRACT: Despite overwhelming evidence of the importance of brain renin-angiotensin system (RAS), the very existence of intrinsic brain RAS remains controversial.
To investigate the hypothesis that the brain (pro)renin receptor (PRR) is physiologically important in the brain RAS regulation and cardiovascular functions.
PRR is broadly distributed within neurons of cardiovascular-relevant brain regions. The physiological functions of PRR were studied in the supraoptic nucleus (SON) because this brain region showed greater levels of PRR mRNA in the spontaneously hypertensive rats (SHR) compared with normotensive Wistar-Kyoto (WKY) rats. Adeno-associated virus (AAV)-mediated overexpression of human PRR in the SON of normal rats resulted in increases in plasma and urine vasopressin, and decreases in H(2)O intake and urine output without any effects on mean arterial pressure and heart rate. Knockdown of endogenous PRR by AAV-short hairpin RNA in the SON of SHRs attenuated age-dependent increases in mean arterial pressure and caused a decrease in heart rate and plasma vasopressin. Incubation of neuronal cells in culture with human prorenin and angiotensinogen resulted in increased generation of angiotensin I and II. Furthermore, renin treatment increased phosphorylation of extracellular signal-regulated kinase ½ in neurons from both WKY rats and SHRs; however, the stimulation was 50% greater in the SHR.
The study demonstrates that brain PRR is functional and plays a role in the neural control of cardiovascular functions. This may help resolve a long-held controversy concerning the existence of intrinsic and functional brain RAS.
Circulation Research 10/2010; 107(7):934-8. · 9.49 Impact Factor
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ABSTRACT: Accumulating evidence indicates a key role of inflammation in hypertension and cardiovascular disorders. However, the role of inflammatory processes in neurogenic hypertension remains to be determined. Thus, our objective in the present study was to test the hypothesis that activation of microglial cells and the generation of proinflammatory cytokines in the paraventricular nucleus (PVN) contribute to neurogenic hypertension. Intracerebroventricular infusion of minocycline, an anti-inflammatory antibiotic, caused a significant attenuation of mean arterial pressure, cardiac hypertrophy, and plasma norepinephrine induced by chronic angiotensin II infusion. This was associated with decreases in the numbers of activated microglia and mRNAs for interleukin (IL) 1beta, IL-6, and tumor necrosis factor-alpha, and an increase in the mRNA for IL-10 in the PVN. Overexpression of IL-10 induced by recombinant adenoassociated virus-mediated gene transfer in the PVN mimicked the antihypertensive effects of minocycline. Furthermore, acute application of a proinflammatory cytokine, IL-1beta, into the left ventricle or the PVN in normal rats resulted in a significant increase in mean arterial pressure. Collectively, this indicates that angiotensin II induced hypertension involves activation of microglia and increases in proinflammatory cytokines in the PVN. These data have significant implications on the development of innovative therapeutic strategies for the control of neurogenic hypertension.
Hypertension 08/2010; 56(2):297-303. · 6.21 Impact Factor
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ABSTRACT: Dysfunction of endothelial nitric oxide synthase (eNOS) has been implicated in the pathogenesis of diabetic vascular complications. This study was undertaken to determine the role of eNOS in the development of diabetic retinopathy (DR), by investigating the functional consequences of its deficiency in the diabetic state.
Diabetes was induced in eNOS-knockout (eNOS(-/-)) and C57B/6 mice by streptozotocin (STZ) injection. Retinal vasculature was evaluated by albumin extravasation, to quantitatively measure vascular permeability, and by trypsin-digested retinal vascular preparations, to quantify acellular capillaries. Gliosis was evaluated by immunofluorescent techniques. Retinal capillary basement membrane thickness was assessed by transmission electron microscopy. Total retinal nitric oxide level was assessed by measuring nitrate/nitrite using a fluorometric-based assay, iNOS expression was examined by real-time PCR.
Diabetic eNOS(-/-) mice exhibit more severe retinal vascular permeability than age-matched diabetic C57BL/6 mice, detectable as early as 3 weeks after diabetes induction. Diabetic eNOS(-/-) mice also show earlier onset and an increased number of acellular capillaries, sustained gliosis, and increased capillary basement membrane thickness. Total nitric oxide (NO) level was also increased, concomitant with elevated iNOS expression in diabetic eNOS(-/-) retina.
Diabetic eNOS(-/-) mice exhibit A significantly wider range of advanced retinal vascular complications than the age-matched diabetic C57BL/6 mice, supporting the notion that eNOS-derived NO plays an essential role in retinal vascular function. This mouse model also faithfully replicates many of the hallmarks of vascular changes associated with human retinopathy, thus providing a unique model to aid in understanding the pathologic mechanisms of and to develop effective therapeutic strategies for diabetic retinopathy.
Investigative ophthalmology & visual science 04/2010; 51(10):5240-6. · 3.43 Impact Factor
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Hypertension 04/2010; 55(6):1308-9. · 6.21 Impact Factor
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ABSTRACT: Achromatopsia is an autosomal recessive retinal disease involving loss of cone function that afflicts approximately 1 in 30,000 individuals. Patients with achromatopsia usually have visual acuities lower than 20/200 because of the central vision loss, photophobia, complete color blindness and reduced cone-mediated electroretinographic (ERG) amplitudes. Mutations in three genes have been found to be the primary causes of achromatopsia, including CNGB3 (beta subunit of the cone cyclic nucleotide-gated cation channel), CNGA3 (alpha subunit of the cone cyclic nucleotide-gated cation channel), and GNAT2 (cone specific alpha subunit of transducin). Naturally occurring mouse models with mutations in Cnga3 (cpfl5 mice) and Gnat2 (cpfl3 mice) were discovered at The Jackson Laboratory. A natural occurring canine model with CNGB3 mutations has also been found. These animal models have many of the central phenotypic features of the corresponding human diseases. Using adeno-associated virus (AAV)-mediated gene therapy, we and others show that cone function can be restored in all three models. These data suggest that human achromatopsia may be a good candidate for corrective gene therapy.
Advances in experimental medicine and biology 01/2010; 664:639-46. · 1.09 Impact Factor
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ABSTRACT: VEGF is recognized as a major mediator in the development of diabetic nephropathy. Soluble Flt-1 (sFlt-1) is the endogenous inhibitor of VEGF, and recently genetic overexpression of sFlt-1 in the podocyte was shown to be protective in murine diabetic nephropathy. In this study, we performed a translational study to determine whether an intramuscular gene transfer of sFlt-1 can prevent the progression of renal disease in diabetic db/db mice. Adeno-associated virus-1 (AAV1) encoding human sFlt-1 in two different doses was intramuscularly administrated in db/db and wild-type mice. The sFlt-1-AAV1 treatment significantly increased serum sFlt-1 level at 4 and 8 wk. A dose that was developed in this study caused minimal abnormalities in normal mice but reduced albuminuria in diabetic db/db mice. In renal histology, sFlt-1 treatment at this dose had minimal effects on mesangial expansion in diabetic mice, whereas podocyte injury was significantly improved, at 8 wk. Unfortunately, tubulointerstitial injury was markedly exacerbated by sFlt-1 treatment in association with a reduction in endogenous VEGF expression and peritubular capillary loss. In conclusion, gene therapy with sFlt-1-AAV1 protects podocytes but accelerates tubulointerstitial injury in diabetic db/db mice. These data suggest systemic overexpression of sFlt-1 will not likely be useful for treating diabetic nephropathy.
AJP Renal Physiology 12/2009; 298(3):F609-16. · 4.42 Impact Factor
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ABSTRACT: Cardiac gene transfer may serve as a novel therapeutic approach for heart disease. Numerous serotypes of recombinant adeno-associated virus (rAAV) have been identified with variable tropisms to cardiac tissue.
Both in vitro and in vivo experiments were undertaken to compare cardiac tropisms of rAAV-2, 5, 7, 8 and 9. For the in vitro studies, 10(7) vector genome (vg) of rAAV-2, 5, 7, 8 or 9 were used to transduce both rat neonatal cardiac myocytes (RNCM) and fibroblasts (RNCF). For the in vivo studies, 4 x 10(10) vg of rAAV-2, 5, 7, 8 or 9, and 4 x 10(11) vg of rAAV8 or 9 were administered in 5-day-old rats via a relatively non-invasive intracardiac injection. One and two months post-administration, green fluorescent protein (GFP) expression in tissues was visualized and GFP mRNA was quantified by the real-time polymerase chain reaction.
At 3 days post-viral transduction, rAAV9 and rAAV2 produced the highest transducing efficiency in RNCM. Only rAAV2 elicited any transduction in the RNCF. The results obtained in vivo indicated that the order for transduction efficiency in the heart was: rAAV9 > rAAV8 > rAAV7 > rAAV2 = rAAV5. The transduction efficiency order in the liver was: rAAV2 > rAAV5 > rAAV7 > rAAV8 > rAAV9. Injection of a higher dose (4 x 10(11) vg) of rAAV9 provided more widespread and highly cardiac-selective GFP expression in the heart than rAAV8. Zero to minimal expression of GFP was found in the lung and kidney for both doses of all rAAV serotypes utilized.
Collectively, the results obtained in the present study suggest that rAAV9 provides the most selective and stable transduction efficiency in cardiac tissue, and this expression was primarily exhibited in cardiac myocytes.
The Journal of Gene Medicine 10/2009; 12(1):22-34. · 2.48 Impact Factor
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ABSTRACT: Red-green colour blindness, which results from the absence of either the long- (L) or the middle- (M) wavelength-sensitive visual photopigments, is the most common single locus genetic disorder. Here we explore the possibility of curing colour blindness using gene therapy in experiments on adult monkeys that had been colour blind since birth. A third type of cone pigment was added to dichromatic retinas, providing the receptoral basis for trichromatic colour vision. This opened a new avenue to explore the requirements for establishing the neural circuits for a new dimension of colour sensation. Classic visual deprivation experiments have led to the expectation that neural connections established during development would not appropriately process an input that was not present from birth. Therefore, it was believed that the treatment of congenital vision disorders would be ineffective unless administered to the very young. However, here we show that the addition of a third opsin in adult red-green colour-deficient primates was sufficient to produce trichromatic colour vision behaviour. Thus, trichromacy can arise from a single addition of a third cone class and it does not require an early developmental process. This provides a positive outlook for the potential of gene therapy to cure adult vision disorders.
Nature 09/2009; 461(7265):784-7. · 36.28 Impact Factor
