Donald D. Lund

Gene Transfer of Extracellular Superoxide Dismutase Reduces Arterial Pressure in Spontaneously Hypertensive Rats: Role of Heparin-Binding Domain

Abstract— Oxidative stress may contribute to hypertension. The goals of this study were to determine whether extracellular superoxide dismutase (ECSOD) reduces arterial pressure in spontaneously hypertensive rats (SHR) and whether its heparin-binding domain (HBD), which is responsible for cellular binding, is necessary for the function of ECSOD. Three days after intravenous injection of an adenoviral vector expressing human ECSOD (AdECSOD), mean arterial pressure (MAP) decreased from 165±4 mm Hg (mean±SE, n=7) to 124±3 mm Hg (n=7) in adult anesthetized SHR (P <0.01) but was not altered in normotensive Wistar-Kyoto rats. Cardiac output was not changed in SHR 3 days after AdECSOD. Gene transfer of ECSOD with deletion of the HBD (AdECSOD&Dgr;HBD) had no effect on SHR MAP, even though plasma SOD activity was greater after AdECSOD&Dgr;HBD than after AdECSOD. Immunohistochemistry revealed intense staining for ECSOD in blood vessels and kidneys after AdECSOD but not after AdECSOD&Dgr;HBD. Impaired relaxation of the carotid artery to acetylcholine in SHR was significantly improved after AdECSOD. Cumulative sodium balance in SHR was reduced by AdECSOD compared with AdECSOD&Dgr;HBD. Gene transfer of ECSOD also reduced MAP in conscious SHR, although the effect was not as profound as in anesthetized SHR. In summary, gene transfer of ECSOD, with a strict requirement for its HBD, reduces systemic vascular resistance and arterial pressure in a genetic model of hypertension. This reduction in arterial pressure may be mediated by vasomotor and/or renal mechanisms.

Gene Transfer of Endothelial Nitric Oxide Synthase Improves Relaxation of Carotid Arteries From Diabetic Rabbits

BACKGROUND Diabetes mellitus is associated with impairment of NO-mediated vascular relaxation. The purpose of this study was to determine whether adenovirus-mediated gene transfer of endothelial NO synthase (eNOS) or Cu/Zn superoxide dismutase (SOD1) improves responsiveness to acetylcholine in alloxan-induced diabetic rabbits. METHODS AND RESULTS After 8 weeks, plasma glucose was greater in diabetic rabbits (418+/-35 mg/dL) (mean+/-SEM) than in normal rabbits (105+/-4 mg/dL). Carotid arteries were removed and cut into ring segments. Arteries were incubated for 2 hours with adenoviral vectors driven by a CMV promoter expressing beta-galactosidase (beta-gal), eNOS, SOD1, or vehicle. After incubation with virus, arteries were incubated for an additional 24 hours to allow transgene expression. Vascular reactivity was examined by recording isometric tension. After precontraction with phenylephrine, responses to the endothelium-independent vasodilator sodium nitroprusside were similar in diabetic and normal arteries. Endothelium-dependent relaxation to acetylcholine (3x10(-6) mol/L) was significantly less in arteries from diabetic animals (68+/-5%) than in normal vessels (90+/-3%). Adenoviral transfection of arteries with eNOS improved relaxation in response to acetylcholine in diabetic (EC(50) eNOS=0.64+/-0.12x10(-7) mol/L versus vehicle =1. 70+/-0.43x10(-7) mol/L) but not normal arteries. Vasorelaxation in response to acetylcholine was inhibited by N(omega)-nitro-L-arginine (100 micromol/L) in all groups. Responses to acetylcholine were unchanged after gene transfection of SOD1 or beta-gal in arteries from diabetic or normal rabbits. CONCLUSIONS Adenovirus-mediated gene transfer of eNOS, but not SOD, improves impaired NO-mediated relaxation in vessels from diabetic rabbits.

Slowing of motor nerve conduction velocity in streptozotocin-induced diabetic rats is preceded by impaired vasodilation in arterioles that overlie the sciatic nerve.

Diabetes mellitus produces marked abnormalities in motor nerve conduction, but the mechanism is not clear. In the present study we hypothesized that in the streptozotocin (STZ)-induced diabetic rat impaired vasodilator function in arterioles that provide circulation to the region of the sciatic nerve is associated with reduced endoneural blood flow (EBF) and that these defects precede slowing of motor nerve conduction velocity, and thereby may contribute to nerve dysfunction. As early as three days after the induction of diabetes endoneural blood flow was reduced in the STZ-induced diabetic rat. Furthermore, after 1 week of diabetes acetylcholine- induced vasodilation was found to be impaired. This was accompanied by an increase in the superoxide level in arterioles that provide circulation to the region of the sciatic nerve as well as changes in the level of other markers of oxidative stress including an increase in serum levels of thiobarbituric acid reactive substances and a decrease in lens glutathione level. In contrast to the vascular related changes that occur within 1 week of diabetes, motor nerve conduction velocity and sciatic nerve Na+/k+ ATPase activity were significantly reduced following 2 and 4 weeks of diabetes, respectively. These studies demonstrate that changes in vascular function in the STZ-induced diabetic rat precede the slowing of motor nerve conduction velocity (MNCV) and are accompanied by an increase in superoxide levels in arterioles that provide circulation to the region of the sciatic nerve.

Organization of the sympathetic postganglionic innervation of the rat heart

The origins and organization of cardiac sympathetic postganglionic nerves in the rat were identified in the present investigation. The retrograde tracer, Diamidino Yellow, was injected into the right or left ventricles to label somata in the sympathetic chain. Analysis of all sympathetic ganglia from superior cervical ganglion through the 10th thoracic ganglion indicated that the postganglionic innervation of the rat cardiac ventricles originates bilaterally. The majority of these somata were located in the middle and inferior cervical ganglia (middle cervical-stellate ganglion complex) (approximately 92% of all labelled cells), with lesser contributions from the superior cervical and 4th through 6th thoracic ganglia. To confirm and further quantitate these findings, the middle cervical-stellate ganglion complex was removed (MC-S ganglionectomy) bilaterally or ipsilaterally from the left or right sides, and regional cardiac norepinephrine concentration (left and right atrial appendages and left and right ventricles) was analysed 7 or 28 days later. At both times after bilateral MC-S ganglionectomy, regional cardiac norepinephrine was reduced by 89% to 100%, indicating the removal of almost all cardiac noradrenergic cells of origin and possibly fibers of passage. The results of unilateral MC-S ganglionectomy experiments indicated that the atrial appendages and the left ventricle receive bilateral innervation from the middle cervical-stellate ganglion complex. However, the left middle cervical-stellate ganglion complex appears to contribute a majority of the norepinephrine to the right ventricle. Furthermore, between 7 and 28 days after contralateral MC-S ganglionectomy, atrial appendages, but not ventricles, display significant recovery of norepinephrine content. The present data demonstrate: (1) a bilateral locus of origin of cardiac sympathetic postganglionic neurons, limited longitudinally to cervical through mid-thoracic ganglia, and (2) the ability of the cardiac postganglionic innervation to regenerate after partial denervation. These results demonstrate anatomical evidence for significant bilateral integration of cardiac sympathetic activity at the level of the sympathetic ganglion in the rat.

Effect of M40403 treatment of diabetic rats on endoneurial blood flow, motor nerve conduction velocity and vascular function of epineurial arterioles of the sciatic nerve

To further explore the effect of antioxidants in preventing diabetes‐induced vascular and neural dysfunction we treated streptozotocin‐induced diabetic rats daily with subcutaneous injections of 10 mg kg−1 of M40403 (n=11) and compared the results obtained from 17 control rats and 14 untreated diabetic rats. M40403 is a manganese(II) complex with a bis(cyclo‐hexylpyridine)‐substituted macrocyclic ligand that was designed to be a selective functional mimetic of superoxide dismutase. Thus, M40403 provides a useful tool to evaluate the roles of superoxide in disease states. Treatment with M40403 significantly improved diabetes‐induced decrease in endoneurial blood flow, acetylcholine‐mediated vascular relaxation in arterioles that provide circulation to the region of the sciatic nerve, and motor nerve conduction velocity (P<0.05). M40403 treatment also reduced the appearance of superoxide in the aorta and epineurial vessels and peroxynitrite in epineurial vessels. Treating diabetic rats with M40403 reduced the diabetes‐induced increase in thiobarbituric acid reactive substances in serum but did not prevent the decrease in lens glutathione level. Treating diabetic rats with M40403 did not improve sciatic nerve Na+/K+ ATPase activity or the sorbitol, fructose or myo‐inositol content of the sciatic nerve. These studies provide additional evidence that diabetes‐induced oxidative stress and the generation of superoxide and perhaps peroxynitrite may be partially responsible for the development of diabetic vascular and neural complications.

Mechanisms of Inducible Nitric Oxide Synthase–Mediated Vascular Dysfunction

Objective—Inducible nitric oxide synthase (iNOS) is expressed in arteries during inflammation and may contribute to vascular dysfunction. Effects of gene transfer of iNOS to carotid arteries were examined in vitro in the absence of systemic inflammation to allow examination of mechanisms by which iNOS impairs contraction and relaxation. Methods and Results—After gene transfer of iNOS with an adenovirus (AdiNOS), constrictor responses to phenylephrine (PE) and U46619 were impaired. After AdiNOS, inhibition of soluble guanylate cyclase (sGC) with 1H-[1,2,4]oxadiazolo-[4,3,2]quinoxalin-1-one (ODQ) reduced the EC50 for PE from 4.33±0.78 &mgr;mol/L to 1.15±0.43 &mgr;mol/L (mean±SEM). These results imply that iNOS impairs contraction by activation of the NO/cGMP pathway. Relaxation to acetylcholine (ACh) also was impaired after AdiNOS. Sepiapterin (300 &mgr;mol/L), the precursor for tetrahydrobiopterin (BH4), improved relaxation to Ach. Because BH4 is an essential cofactor for production of NO by both iNOS and endothelial nitric oxide synthase (eNOS), these results suggest that iNOS may reduce production of NO by eNOS by limiting availability of BH4. Next, we examined effects of expression of iNOS in endothelium and adventitia. Selective expression of iNOS in endothelium, but not adventitia, impaired contraction to phenylephrine and relaxation to acetylcholine. Conclusions—We conclude that: (1) iNOS may impair contraction in part by activation of sGC; (2) iNOS impairs relaxation, at least in part, by limiting availability of BH4; and (3) expression of iNOS in endothelium may be a more important mediator of vascular dysfunction than expression of iNOS in adventitia.

Location, distribution and projections of intracardiac ganglion cells in the rat

Physiological studies indicate that cardiac parasympathetic nerves may act selectively at discrete cardiac sites. To determine anatomical sites at which selective integration of cardiac nerve activity may occur, the present study identified and described the location, distribution, and projections of intracardiac ganglion cells in the rat. The estimated 3992 ganglion cells per rat heart were located in 4 distinct groups, all above the atrioventricular groove: (1) between the superior vena cava and aorta (2.5% of total), (2) in the region of the superior interatrial septum (49.9%), (3) posterior to the left atrium (24.0%), and (4) posterior to the inferior interatrial septum and right atrium (23.5%). Only a few ganglion cells were located subepicardially within the infolding of the dorsal interatrial septum. Retrogradely transported fluorescent tracers injected into the left or right ventricles demonstrated that different groups of ganglion cells projected to discrete or selective regions of the heart. Projections to the left ventricle originate only from ganglion cells located posterior to the interatrial septum and the left atrium. In the rat, intracardiac ganglion cells, confined to 4 atrial regions, appear to have discrete sites of termination within the heart. It is proposed that selective activation of different intracardiac ganglion cell groups may elicit specific regional changes in cardiac parasympathetic nerve activity.

Vascular Effects of the Human Extracellular Superoxide Dismutase R213G Variant

Background—Extracellular superoxide dismutase (ECSOD) is a major extracellular antioxidant enzyme. We have demonstrated that vascular effects of ECSOD require an intact heparin-binding domain. A common genetic variant with a substitution in the heparin-binding domain (ECSODR213G) was reported recently to be associated with ischemic heart disease. The goal of this study was to examine vascular effects of ECSODR213G. Methods and Results—A recombinant adenovirus (Ad) that expresses ECSODR213G was constructed. ECSODR213G and ECSOD proteins bound to collagen type I in vitro, but binding to aorta ex vivo was 10-fold greater with ECSOD than ECSODR213G. Three days after intravenous injection of AdECSODR213G or AdECSOD in spontaneously hypertensive rats (SHR), immunostaining demonstrated binding of ECSOD to carotid arteries and kidneys but minimal binding of ECSODR213G. Binding to aorta and carotid artery was 2.5- to 3-fold greater with ECSOD than ECSODR213G by immunoblotting. Arterial pressure was significantly reduced by AdECSOD but not by AdECSODR213G. Responses to acetylcholine and basal levels of nitric oxide in carotid arteries were impaired in SHR compared with normotensive Wistar-Kyoto rats and were improved after AdECSOD but not AdECSODR213G. Levels of superoxide and nitrotyrosine in aorta were higher in SHR than Wistar-Kyoto rats and were greatly reduced after AdECSOD but not AdECSODR213G. Conclusions—In contrast to ECSOD, ECSODR213G has no significant protective effect on arterial pressure, vascular function, or vascular levels of oxidative stress in SHR. These findings may provide a mechanistic basis for association studies that suggest that human beings carrying ECSODR213G are predisposed to vascular diseases.

Alterations in the baroreceptor reflex control of heart rate in streptozotocin diabetic rats.

Baroreflex control of heart rate was studied in conscious diabetic rats at 12, 24 and 48 weeks after the induction of diabetes with streptozotocin. Baseline blood pressure (mean arterial blood pressure) of diabetic rats was significantly lower at 12 weeks after the induction of diabetes when compared to age-matched control rats. However, at 24 and 48 weeks of diabetes, no difference in blood pressure was observed between diabetic and age-matched control rats. In contrast, bradycardia (prolongation of pulse interval) was a consistent feature of diabetic rats at all time points (12, 24, and 48 weeks). To assess parasympathetic control of heart rate, baroreceptor sensitivity was determined by infusing phenylephrine. Baroreflexes in diabetic rats were changed from an increased sensitivity at 12 and 24 weeks to decreased sensitivity at 48 weeks after the induction of diabetes. This suggests that alterations in baroreflex sensitivity might depend upon the length of time the animals were exposed to the diabetes. Insulin treatment in diabetic animals reversed hypotension, bradycardia and altered baroreflex sensitivity observed in 12-week diabetic rats. Non-diabetic rats, in which the development of diabetes was prevented by pretreatment with 3-0-methylglucose before streptozotocin injection, or rats which did not develop diabetes after streptozotocin injection showed a similar baseline blood pressure, heart rate and baroreflex sensitivity to those of age-matched control rats (12, 24 and 48 weeks). This data suggests that changes in blood pressure, heart rate and baroreflex sensitivity are due to the diabetic state, not to streptozotocin toxicity.

Attenuation of Vascular/Neural Dysfunction in Zucker Rats Treated With Enalapril or Rosuvastatin

Objective: Obese Zucker rats, animal model for the metabolic syndrome, develop a diabetes‐like neuropathy that is independent of hyperglycemia. The purpose of this study was to determine whether drugs used to treat cardiovascular dysfunction in metabolic syndrome also protect nerve function.