Bingyin Wang

Overexpression of Dimethylarginine Dimethylaminohydrolase Reduces Tissue Asymmetric Dimethylarginine Levels and Enhances Angiogenesis

Background—This study was designed to determine whether overexpression of the enzyme dimethylarginine dimethylaminohydrolase (DDAH) could enhance angiogenesis by reducing levels of the endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA). Methods and Results—In DDAH1 transgenic (TG) and wild-type mice (each n=42), the role of DDAH overexpression on angiogenesis was studied by use of the disk angiogenesis system and a murine model of hindlimb ischemia (each n=21). After surgery, animals were treated with either PBS or the NOS inhibitors ADMA or N&ohgr;-nitro-l-arginine methyl ester (L-NAME; each 250 &mgr;mol · kg−1 · d−1) by use of osmotic minipumps (each n=7). L-NAME was chosen to study an inhibitor that is not degraded by DDAH. Neovascularization in the disk angiogenesis system was impaired by both NOS inhibitors; however, TG animals were resistant to the effects of ADMA on neovascularization. Similarly, TG mice were more resistant to the inhibitory effect of ADMA on angioadaptation (angiogenesis and arteriogenesis) after hindlimb ischemia, as assessed by fluorescent microsphere studies and postmortem microangiograms. Enhanced neovascularization and limb perfusion in TG mice were associated with reduced plasma and tissue ADMA levels and enhanced tissue NOS enzyme activity. Conclusions—We describe a novel mechanism by which DDAH regulates postnatal neovascularization. Therapeutic manipulation of DDAH expression or activity may represent a novel approach to restore tissue perfusion.

Local Intramural Delivery of l-Arginine Enhances Nitric Oxide Generation and Inhibits Lesion Formation After Balloon Angioplasty

BACKGROUND Long-term oral administration of L-arginine has been shown to enhance production of nitric oxide (NO) and to reduce lesion formation. The goal of this study was to determine whether local intramural administration of L-arginine could enhance NO generation and reduce intimal thickening. METHODS AND RESULTS New Zealand White rabbits (n = 27) received a 1% cholesterol diet. For the short-term study, after 1 week of diet, both iliac arteries were balloon injured. Four weeks later, vasoreactivity was assessed angiographically during infusion of acetylcholine (Ach) before and after delivery of L-arginine or saline into the right or left iliac artery (800 mg/5 mL; 0.2 mL/min, 15 minutes) by use of a local drug-delivery balloon. Vessels were then harvested for measurements of NO. For the long-term study, after balloon injury, drugs were delivered as above into the iliac arteries. Two and 4 weeks after L-arginine delivery, vasoreactivity was determined. Subsequently, the iliac arteries were harvested for histomorphometric analysis and measurements of NO. In the short-term study, local delivery of L-arginine restored endothelium-dependent vasodilatation (Ach 10(-5) mol/L; L-arginine +35 +/- 10%; saline -14 +/- 5%; P < .001) and enhanced local production of nitrogen oxides (L-arginine 152 +/- 28; saline 78 +/- 12 nmol/L per milligram of tissue per hour; P < .04). In the long-term study, local administration of L-arginine enhanced vascular NO production as long as 1 week after the injury (L-arginine 394.4 +/- 141.6; saline 86.3 +/- 34.3 nmol/L per milligram of tissue per hour; P < .01) and reduced intimal thickening 4 weeks later (intima/ media ratio: L-arginine 0.56 +/- 0.1; saline 1.40 +/- 0.2; P < .001), largely due to suppression of macrophage accumulation. CONCLUSIONS A single intramural administration of L-arginine enhances vascular NO generation and inhibits lesion formation. Local augmentation of NO production at the site of balloon angioplasty may be a novel strategy to prevent restenosis.

Local l-Arginine Delivery After Balloon Angioplasty Reduces Monocyte Binding and Induces Apoptosis

BACKGROUND Local administration of L-arginine after balloon angioplasty has been shown to enhance NO generation and inhibit lesion formation. In this study, we assessed the mechanisms by which local delivery of L-arginine inhibits lesion formation. METHODS AND RESULTS New Zealand White rabbits (n=56) were fed a 1% cholesterol diet. After 1 week, both iliac arteries were balloon-denuded, and a local drug delivery catheter was introduced into both iliac arteries to deliver either L-arginine (800 mg/5 mL with and without 100 microCi L-[2,3-(3)H]-arginine) or saline. Monocyte-endothelial interaction was assessed by functional binding assay; NO activity was measured by chemiluminescence. Intramural administration of radioactively labeled L-arginine led to significantly higher counts in comparison to the contralateral segment for up to 1 week after delivery (676+/-223 versus 453+/-93 cpm/mg; P<0.02); this was associated with significantly higher NO levels in the L-arginine-treated segments (394.4+/-141.6 versus 86.3+/-34.3 nmol/mg; P<0.01). Even after 2 to 3 weeks, monocyte binding was significantly decreased by treatment with L-arginine as compared with saline infusion (P<0.01). After 4 weeks, there was a 9-fold greater number of apoptotic cells in the vessel wall of L-arginine as compared with the saline-treated segments (P<0.05). CONCLUSIONS Intramural delivery of L-arginine immediately after angioplasty causes a sustained increase in tissue L-arginine levels associated with enhancement of local NO synthesis. The local increase in NO synthesis is associated with an attenuation of monocyte binding and increased apoptosis of resident macrophages. This treatment strategy could be valuable for the prevention and management of restenosis.

Interaction of Diabetes and Hypertension on Determinants of Endothelial Adhesiveness

Epidemiological studies have established that diabetes mellitus and hypertension are independent risk factors for atherosclerosis. One of the earliest abnormalities seen in atherogenesis is enhanced monocyte adherence to the endothelium. The mechanisms by which diabetes mellitus or hypertension enhances monocyte-endothelial cell interactions are incompletely characterized. It is not known whether there are additive interactions between these risk factors on endothelial adhesiveness for monocytes. Male Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats were fed a normal or fructose-enriched diet. In some cases, animals were injected with streptozotocin (35 mg/kg body weight) to induce diabetes. After 2 weeks, plasma was drawn for biochemical measurements, and thoracic aortas were harvested, opened longitudinally, and exposed to fluorescently labeled mouse monocytoid cells (WEHI 78/24, 2 x 10(6)/mL) for 30 minutes on a rocking platform. Adherent cells were counted by epifluorescence microscopy. WEHI 78/24 binding to aortic segments from SHR animals was elevated compared with segments from WKYs. Fructose feeding alone had no effect on endothelial adhesiveness. When WKYs were made hyperglycemic by STZ injection, monocyte binding was 160% of the control value. Elevated monocyte binding was also observed in aortas derived from SHR animals injected with STZ, indicating an additive effect of hypertension and hyperglycemia. To determine whether alterations in oxidative state played a role in the endothelial adhesiveness, aortic segments were exposed to lucigenin (250 micromol/L) for measurement of superoxide anion. Aortic segments from SHR elaborated 120% more superoxide anion than did controls. Elevated free-radical production was also observed in aortas from diabetic WKYs. Furthermore, thoracic aortas derived from diabetic SHR animals elaborated more superoxide anion than did any of the other groups (374%, P<0.05). Immunohistochemical staining for monocyte chemotactic protein-1 demonstrated increased expression in aortas isolated from diabetic WKY and SHR compared with control vessels. These studies demonstrate that both diabetes and hypertension lead to increased monocyte adherence to the endothelium. This abnormality is associated with increased vascular superoxide production and monocyte chemotactic protein-1 expression. Furthermore, there appears to be an additive interaction between hyperglycemia and hypertension in their effects on endothelial adhesiveness and its determinants.

Discordant effects of dietary L-arginine on vascular structure and reactivity in hypercholesterolemic rabbits

We investigated the effect of dietary supplementation of L-arginine (L-Arg), the precursor of endothelial nitric oxide (NO), on endothelium-dependent and endothelium-independent vascular responses, as well as vascular structure, in the abdominal aorta of hypercholesterolemic rabbits. Rabbits were fed (a) normal rabbit chow, (b) 1% cholesterol diet, or (c) 1% cholesterol diet supplemented with 2.25% L-Arg HCl in drinking water. After 10 weeks, the abdominal aorta was harvested for study of vascular reactivity and histomorphometry. L-Arg did not affect serum cholesterol levels. Histomorphometric analysis demonstrated an eightfold reduction in intimal thickening in the abdominal aorta of the arginine-supplemented hypercholesterolemic rabbits. By contrast, the effects on vascular reactivity were subtle. Contraction to norepinephrine (NE) was not altered by hypercholesterolemia or L-Arg. Contraction to acetylcholine (ACh) was increased in hypercholesterolemic animals; this was normalized by dietary arginine supplementation. Relaxation to nitroglycerin (NTG) was not altered by hypercholesterolemia but was attenuated in the arginine-supplemented rabbits. Endothelium-dependent relaxation to ACh was impaired in both hypercholesterolemic groups. Dietary L-Arg has a dramatic antiatherogenic effect in hypercholesterolemic rabbits. This effect is associated with rather slight changes in vascular reactivity that are suggestive of a slight increase in NO elaboration by the endothelium. The discordance between the effects of dietary arginine on vascular structure and reactivity suggests that the antiatherogenic effects of the NO precursor may not be mediated entirely by its effect on the endothelium.

Structural changes in the bladder walls of pregnant and hormone-treated rats: correlation with bladder dynamics

To evaluate the effects of oestrogen, progesterone and pregnancy on bladder dynamics, and assess the associated histological and structural changes in the bladder wall in a rat model.

Cholinergic activation of hematopoietic stem cells: role in tobacco-related disease?

Tobacco use is associated with an increase in the white blood cell (WBC) count. This association has been attributed to bronchopulmonary inflammation and/or infection. It is not known if nicotine itself may play a role. The objective of this study was to determine whether nicotine itself could affect the WBC count, and to determine whether this was due to a direct effect on hematopoietic stem cells (HSC). C57Bl6J mice received nicotine orally, and measurements of the WBC count, bone marrow and spleen cellularity, and HSC count were made. To determine the functionality of HSCs, irradiated animals received bone marrow transplants from vehicle or nicotine-treated mice. Nicotine increased leukocytes in the peripheral blood, bone marrow and spleen. The peripheral red cell and platelet count were unaffected. Nicotine increased the frequency of HSC in the bone marrow. Isolated long-term HSCs from nicotine-treated mice transplanted into irradiated mice regenerated all hematopoietic cell lineages, demonstrating the functional competence of those HSCs. HSCs expressed nicotinic acetylcholine receptors (nAChRs), as documented by FITC-conjugated alpha-bungarotoxin binding. Nicotine increased soluble Kit ligand, consistent with stem cell activation. In conclusion, the data suggest a new mechanism for the increased WBC associated with tobacco use. The effect of nicotine to activate hematopoiesis may contribute to tobacco-related diseases.

The effects of intravesical lidocaine on bladder dynamics of children with myelomeningocele.

PURPOSE Other studies have suggested that intravesical lidocaine may temporarily improve bladder dynamics but details of these effects and their application to children have not been examined. We evaluated the effects of intravesical lidocaine on bladder urodynamics of children with myelomeningocele and tried to correlate these effects with subsequent clinical response to oral oxybutynin. MATERIALS AND METHODS Charts of children with myelomeningocele who had undergone urodynamic examinations from 1992 to 1998 were reviewed retrospectively. In children with uninhibited contractions or poor compliance 150 to 300 mg. lidocaine were instilled for 8 minutes and cystometry was repeated. Changes in bladder capacity and compliance, number of uninhibited contractions and bladder volume at which pressure of 40 cm. H2O was reached were recorded before and after the lidocaine instillation. Clinical response to subsequent treatment with oral oxybutynin was assessed from chart review. RESULTS A total of 48 urodynamic studies in 22 girls and 20 boys with a mean age plus or minus standard deviation of 8.3 +/- 5.7 years and myelomeningocele were evaluable. After instillation of lidocaine, urodynamics showed increased bladder capacity in 70.8% of studies (34 of 48), with an average increase in volume of 66% (p <0.05). No change or decreased bladder capacity occurred in 29.2% of studies. Bladder compliance improved in 61.7% of the studies (29 of 47, p <0.05) and worsened in 38.3%. Bladder volume at which the pressure of 40 cm. H2O was reached increased in 77.8% of studies (14 of 18, p <0.05). After lidocaine the number of uninhibited contractions decreased by 3.2 in 56.8% of studies (21 of 37, p <0.05). Correlation of lidocaine induced changes in bladder capacity, compliance and number of uninhibited contractions with improvement on oral oxybutynin was 70.6%, 64.3% and 66.7%, respectively. CONCLUSIONS Intravesical lidocaine can improve bladder capacity and compliance and decrease the number of uninhibited contractions in many children with neurogenic bladder caused by myelomeningocele. These observations suggest that intravesical lidocaine has effects on the neurogenic bladder that improve bladder dynamics. Although intravesical lidocaine testing may not reliably predict clinical response to oral oxybutynin at the prescribed dosages, a possible therapeutic role for intravesical lidocaine or similar agents should be explored further.

NOx and ADMA changes with focal ischemia, amelioration with the chaperonin GroEL

Both nitric oxide and asymmetric dimethylarginine (ADMA) play a critical role in the regulation of cerebral blood flow, though their neuroprotective and cytotoxic effects are still under investigation. In this study, we found that nitrate/nitrite (NOx) levels in plasma, ischemic brain tissue, and cerebrospinal fluid (CSF) increased significantly 24h after 2h transient middle cerebral artery occlusion (MCAO) in rats. ADMA levels were unchanged in plasma, but decreased significantly in CSF 24h following MCAO. The CSF ADMA/NOx ratio decreased markedly following ischemia. Rats protected by expression of the chaperonin GroEL or its folding deficient mutant D87K had lower plasma NOx levels at 24h reperfusion. ADMA, NO, and their ratio in CSF merit further study as biomarkers for ischemic brain injury.

Testosterone Changes Bladder and Kidney Structure in Juvenile Male Rats

PURPOSE Testosterone affects male development, maturation and aging but limited data exist on testosterone effects on the juvenile genitourinary system. We hypothesized that testosterone has bladder and kidney developmental effects, and investigated this in juvenile male rats. MATERIALS AND METHODS To examine the testosterone effect 21-day-old prepubertal male Wistar rats were divided into 3 groups of 12 each, including sham orchiectomy as controls, and bilateral orchiectomy with vehicle and bilateral orchiectomy with testosterone. Starting at age 28 days (week 0) testosterone enanthate (5 mg/100 gm) or vehicle was injected weekly. Testosterone was measured at study week 0 before injection, and at weeks 1, 6 and 16. Whole bladders and kidneys were evaluated for androgen receptor, bladder collagen-to-smooth muscle ratio, and renal morphometry and immunohistochemistry. RESULTS Testosterone was not detectable at week 0 in all groups. It remained undetectable at weeks 1, 6 and 16 in the orchiectomy plus vehicle group. Testosterone levels were physiological in controls and rats with orchiectomy plus testosterone but levels were higher in the latter than in the former group. Rats with orchiectomy plus testosterone had increased bladder-to-body and kidney-to-body weight ratios (p<0.01 and <0.05, respectively), and decreased collagen-to-smooth muscle ratio than the orchiectomy plus vehicle and control groups. Rats with orchiectomy plus testosterone had a lower renal total glomerular count (p<0.01) but increased androgen receptor density. CONCLUSIONS In juvenile male rats testosterone was associated with increased bladder and renal mass, and increased bladder smooth muscle. Testosterone associated kidneys also appeared to have fewer but larger glomeruli. These data support an important role for sex hormones in structural and functional development of the bladder and kidney.