Vascular remodeling is regulated by a combination of hemodynamic, environmental, and genetic factors and may be influenced by age. To evaluate age-dependent remodeling in rats, we developed and used a quantitative highly reproducible model of carotid flow alteration. Fourteen juvenile (99+/-3 g) and 9 adult (199+/-5 g) male inbred Fischer rats underwent ligation of the left internal and external carotid arteries under anesthesia. Left common carotid blood flow immediately decreased by approximately 93%, whereas flow in the contralateral carotid increased by approximately 46%. After 4 weeks, the left carotid outer diameter (OD) significantly decreased in both juvenile and adult rats (as measured in vivo and by histological morphometry) compared with sham-operated rats. Changes in shear stress acutely mirrored the changes in blood flow. OD increased and shear stress returned to initial values after chronic exposure to increased flow in juvenile but not adult rats. To develop a simple quantitative index of remodeling that would not require killing the animals, we measured the OD in vivo and compared the ratio of right to left OD (OD ratio [ODR]) between groups. The initial ODR for all groups was approximately 1.0. After 4 weeks of altered flow, the ODR was significantly greater in juvenile than in adult rats (1.48+/-0.05 versus 1.29+/-0.04, respectively; P=.030), indicating that juvenile rats experienced more extensive remodeling than did the adult rats. We also found that unilateral carotid ligation caused a left versus right difference in endothelial NO synthase protein levels after 4 weeks that was not present in the sham-operated animals. Thus, the model described here shows that flow-induced vascular remodeling is dependent on age and supports the hypothesis that the driving force for remodeling involves shear stress and possibly NO. Because the model is quantitative, it allows dissection of the genetic factors that regulate remodeling in inbred rat strains.

Blood flow and the tractive force shear stress are important determinants of artery caliber, and reduced shear predisposes arteries to intimal thickening and atherosclerosis. The molecular basis for shear-induced changes in artery wall structure is poorly defined. A number of factors associated with normal and pathological artery wall remodeling are induced by shear stress in endothelial cell cultures. These include platelet-derived growth factor (PDGF), a potent mitogen, chemoattractant, and vasoconstrictor. To determine whether similar changes occur in vivo, we examined the effects of reduced blood flow on endothelial cell PDGF expression and proliferation in the rat carotid artery. Branches of the right internal and external carotid arteries were ligated, reducing common carotid artery blood flow from 8.0+/-0.6 to 0.5+/-0.1 mL/min while increasing flow in the left carotid from 7.1+/-0.6 to 10.8+/-0.7 mL/min. Shear stress following the procedure was 1.4+/-0.2 and 33.4+/-1.1 dyne/cm2 in carotids with reduced blood flow (RF) and increased blood flow (IF), respectively. Arteries were harvested 6, 24, 48, or 72 hours after ligation, perfusion-fixed, and opened longitudinally. Endothelial cell proliferation (bromodeoxyuridine [BrdU] labeling) was assessed en face at 24, 48, and 72 hours; expression of mRNA for PDGF-A and -B chains and PDGF alpha- and beta-receptors (in situ hybridization) was determined at 6, 48, and 72 hours after unilateral flow reduction. RF induced endothelial cell proliferation, which peaked at 48 hours (RF BrdU labeling: 24 hours, 0.4+/-0.2%; 48 hours, 7.2+/-2.0%; and 72 hours, 4.1+/-0.6%; n=5). PDGF-B expression increased in RF compared with IF endothelium within 48 hours and persisted at 72 hours (percent labeling [RF/IFx100]: 6 hours, 76+/-20%; 48 hours, 395+/-179%; and 72 hours, 208+/-44%; n=3). PDGF-A expression was similarly increased in RF endothelium. In contrast, expression of PDGF alpha- and beta-receptors was undetectable in RF and IF endothelium at all times. We conclude that endothelial cell PDGF ligand expression is induced by reduced shear stress in vivo and may play an important role in flow-mediated remodeling and atherogenesis.

Vasodilation following the infusion of acetylcholine is due to the release of endothelium-derived relaxing factor (EDRF). However, the role of EDRF in neurogenic coronary vasodilation, when acetylcholine is released outside the vessel at the adventitial-medial junction, has not been established. The action of EDRF in parasympathetic coronary vasodilation was tested in the present study using a specific inhibitor of EDRF synthesis, nitro-L-arginine methyl ester (L-NAME). Experiments were conducted on closed-chest, alpha-chloralose-anesthetized dogs with the heart paced at a constant rate. Phentolamine and propranolol were administered to block alpha- and beta-adrenergic receptors, and ibuprofen was given to inhibit prostaglandin synthesis. Intracoronary infusion of L-NAME decreased the coronary vasodilation in response to intracoronary acetylcholine or vagal stimulation. The coronary response to the endothelium-independent vasodilator nitroglycerin was unaffected by L-NAME. These data demonstrate that L-NAME specifically inhibits coronary vasodilation caused by acetylcholine and vagal stimulation, indicating that parasympathetic coronary vasodilation is dependent on EDRF.

It is usually assumed that the increase in coronary blood flow observed with norepinephrine occurs through local metabolic vasodilation secondary to cardiac beta-receptor activation. However, direct feedforward beta-receptor-mediated coronary vasodilation is also a possibility. In dogs with alpha-receptor blockade, the left circumflex artery was perfused at constant pressure. The vasodilator effect of intracoronary norepinephrine injections was determined during prolonged diastoles to avoid the chronotropic and intropic effects of norepinephrine. Norepinephrine caused a dose-dependent increase in coronary blood flow that was attenuated by both the selective beta 1-antagonist practolol and the selective beta 2-antagonist ICI 118,551. These data indicate that norepinephrine activates beta 1- and beta 2-receptors in coronary resistance vessels to cause vasodilation independent of inotropic and chronotropic effects. The physiological significance of coronary beta-receptor-mediated vasodilation was investigated in the beating heart. The coronary blood flow response and coronary venous oxygen tension response were compared when myocardial oxygen consumption was increased over the same range by one of three positive inotropic interventions: (1) norepinephrine, (2) paired-pulse stimulation, or (3) norepinephrine after alpha-blockade. During norepinephrine infusion (intervention 1), coronary venous oxygen tension decreased, indicating that the match between myocardial oxygen consumption and oxygen delivery is not maintained when coronary blood flow is controlled by alpha- and beta-receptors in addition to local metabolic factors. Paired-pulse stimulation (intervention 2) also resulted in a decrease in coronary venous oxygen tension, demonstrating that the balance between oxygen consumption and delivery is not maintained when blood flow is controlled by local metabolic factors alone. However, when coronary beta-receptor-mediated vasodilation was unmasked by alpha-blockade, norepinephrine infusion (intervention 3) produced no change in coronary venous oxygen tension. Therefore, coronary beta-receptor vasodilation helps maintain the balance between flow and metabolism in a feedforward manner in the beating heart.

Abstract-Endothelial nitric oxide synthase (eNOS) has been shown to be regulated both transcriptionally and posttranslationally in cultured endothelial cells, but eNOS regulatory mechanisms in vivo have not been elucidated. Because one of the strongest stimuli for eNOS expression in tissue culture is cell proliferation and because increased NO production would be beneficial in the setting of arterial injury, we hypothesized that eNOS expression should be increased in regenerating endothelium after a denuding injury. Rat aortas underwent partial endothelial denudation by passage of a deflated balloon catheter, and eNOS expression was studied 48 hours after injury. Immunohistochemistry with eNOS monoclonal antibody, NADPH diaphorase activity assay under conditions specific for eNOS, and mRNA hybridization were performed in situ on perfusion-fixed rat aortic segments. The vessels were studied en face to enhance visualization compared with cross sections. eNOS protein and mRNA expression were significantly increased in regenerating and migrating endothelial cells at the wound edge, with translocation of eNOS to the plasma membrane at the leading edge. Similar results were obtained when endothelial cells were studied in a tissue culture wound model. An important role for transforming growth factor (TGF)-␤ 1 in regulating eNOS expression was suggested by the ability of a TGF-␤ 1 -neutralizing antibody to limit induction of eNOS at the wound edge. Increased eNOS expression after wounding appears to be related to signal events associated with cell migration as well as proliferation, because eNOS expression in vivo increased in nonproliferating cells and TGF-␤ 1 -neutralizing antibody inhibited eNOS expression but stimulated proliferation. The current study is the first to suggest an important role in vivo for increased eNOS, and perhaps NO production, in the process of endothelial regeneration and wound repair. (Arterioscler Thromb Vasc Biol. 1998;18:1312-1321.)

Abstract-An important compensatory response to atherosclerosis is vascular remodeling, with maintenance of vessel lumen diameter and shear stress. Both hemodynamic and environmental factors contribute to vascular remodeling and shear stress regulation, and the process is probably also influenced by genetic factors. To establish an animal model for genetic analysis of shear stress regulation and vascular remodeling, we studied the effects of chronic flow alteration in four inbred rat strains. By ligating the left internal and external carotid arteries, we caused a Ϸ90% decrease in left common carotid blood flow and a Ϸ50% increase in right (contralateral) common carotid flow. After 4 weeks of altered flow, there were significant interstrain differences with respect to the change in carotid outer diameter (OD), the relationship between flow and shear stress, and the extent to which shear stress was normalized. Genetically hypertensive rats (GH) exhibited the greatest reduction in shear stress in response to increased flow, stroke-prone spontaneously hypertensive rats (SHR-SP) exhibited a smaller response, and Brown Norway (BN) rats exhibited the smallest response. SHR-SP and GH also differed significantly in outward remodeling (defined as an increase in lumen and vessel diameter) in increased flow arteries. In response to decreased flow, BN rats exhibited the smallest reduction in shear stress. These findings demonstrate significant strain-dependent differences in shear stress regulation and vascular remodeling in response to altered flow. This study emphasizes the important role of genetic factors in vascular remodeling and suggests that genetic analysis of these strains will provide novel insights into the underlying mechanisms. Key Words: carotid artery Ⅲ genetics Ⅲ polymorphism Ⅲ shear stress Ⅲ inbred rat strains T reatment of chronic vascular disease has largely focused on reducing intimal mass to maintain adequate lumen size. 1 However, Glagov 2 demonstrated in 1987 that intimal mass is not the primary factor that determines lumen size in human coronary arteries because vessels remodel to accommodate increasing plaque burden. More recently, intravascular ultrasound (IVUS) has confirmed that vessels can either enlarge (remodel outward) or constrict (remodel inward) at sites of atherosclerosis and after balloon angioplasty. 3,4 These observations suggest that vascular remodeling is an important determinant of vessel lumen size and may alter the clinical course of atherosclerosis.Despite the clinical significance of this process, the mechanisms responsible for vascular remodeling are largely unknown. An important stimulus for vascular remodeling is likely to be blood flow. As blood flows along a vessel, it creates shear stress on the vessel wall. It has been well established that the development of atherosclerosis is related to shear stress with an increased predilection for atherosclerosis to develop in regions of low shear stress and/or turbulence. 5 Endothelial cells may act as the sensors and mediators of t...

1 The functional role of the nitric oxide (NO)/guanosine 3':5'-cyclic monophosphate (cyclic GMP) pathway in experimental myocardial ischaemia and reperfusion was studied in rat isolated hearts. 2 Rat isolated hearts were perfused at constant pressure with Krebs-Henseleit bu er for 25 min (baseline), then made ischaemic by reducing coronary¯ow to 0.2 ml min 71 for 25 or 40 min, and reperfused at constant pressure for 25 min. Drugs inhibiting or stimulating the NO/cyclic GMP pathway were infused during the ischaemic phase only. Ischaemic contracture, myocardial cyclic GMP and cyclic AMP levels during ischaemia, and recovery of reperfusion mechanical function were monitored. 3 At baseline, heart rate was 287+12 beats min 71 , coronary¯ow was 12.8+0.6 ml min 71 , left ventricular developed pressure (LVDevP) was 105+4 mmHg and left ventricular end-diastolic pressure 4.6+0.2 mmHg in vehicle-treated hearts (control; n=12). Baseline values were similar in all treatment groups (P40.05). 4 In normoxic perfused hearts, 1 mM N G -nitro-L-arginine (L-NOARG) signi®cantly reduced coronarȳ ow from 13.5+0.2 to 12.1+0.1 ml min 71 (10%) and LVDevP from 97+1 to 92+1 mmHg (5%; P50.05, n=5). 5 Ischaemic contracture was 46+2 mmHg, i.e. 44% of LVDevP in control hearts (n=12), una ected by low concentrations of nitroprusside (1 and 10 mM) but reduced to *30 mmHg (*25%) at higher concentrations (100 or 1000 mM; P50.05 vs control, n=6). Conversely, the NO synthase inhibitor L-NOARG reduced contracture at 1 mM to 26+3 mmHg (23%), but increased it to 63+4 mmHg (59%) at 1000 mM (n=6). Dobutamine (10 mM) exacerbated ischaemic contracture (81+3 mmHg; n=7) and the cyclic GMP analogue Sp-8-(4-p-chlorophenylthio)-3',5'-monophosphorothioate (Sp-8-pCPT-cGMPS; 10 mM) blocked this e ect (63+1 mmHg; P50.05 vs dobutamine alone, n=5). 6 At the end of reperfusion, LVDevP was 58+5 mmHg, i.e. 55% of pre-ischaemic value in control hearts, signi®cantly increased to *80% by high concentrations of nitroprusside (100 or 1000 mM) or L-NOARG at 1 mM, while a high concentration of L-NOARG (1000 mM) reduced LVDevP to *35% (P50.05 vs control; n=6). 7 Ischaemia increased tissue cyclic GMP levels 1.8 fold in control hearts (P50.05; n=12); nitroprusside at 1 mM had no sustained e ect, but increased cyclic GMP *6 fold at 1000 mM; L-NOARG (1 or 1000 mM) was without e ect (n=6). Nitroprusside (1 or 1000 mM) marginally increased cyclic AMP levels whereas NO synthase inhibitors had no e ect (n=6). 8 In conclusion, the cardioprotective e ect of NO donors, but not of low concentrations of NO synthase inhibitors may be due to their ability to elevate cyclic GMP levels. Because myocardial cyclic GMP levels were not a ected by low concentrations of NO synthase inhibitors, their bene®cial e ect on ischaemic and reperfusion function is probably not accompanied by reduced formation of NO and peroxynitrite in this model.

Recent evidence shows that norepinephrine affects coronary blood flow not only by alpha-receptor-mediated vasoconstriction and by feedback metabolic vasodilation that occurs as a result of myocardial beta-receptor activation, but also by the direct activation of coronary vascular beta-receptors that increase flow in a feedforward manner. The implications of combined feedforward and feedback control in maintaining the balance between metabolism and flow were investigated in the present mass balance model. Feedback was represented by a closed loop and was based on the hypothesis that the regulated variables are myocardial PO2 and PCO2 and that divergence of these variables from their operating point values functions as the metabolic error signals that manipulate coronary vascular smooth muscle and flow to match metabolism. alpha-Receptor-mediated vasoconstriction and beta-receptor-mediated vasodilation are represented by feedforward open loops that are activated simultaneously with increases in metabolism. The postulated control schemes of 1) metabolic feedback control alone, 2) feedback plus alpha- and beta-adrenergic feedforward control, and 3) feedback plus beta-adrenergic feedforward control were able to simulate the steady-state increase in coronary flow and the decrease in coronary venous PO2 that occurs during comparable experimental conditions. The simulations demonstrate that 1) the speed and accuracy of the flow response improve as beta-adrenergic feedforward control is added and alpha-adrenergic feedforward control is removed from the control scheme, 2) high feedback gain also improves the accuracy of the flow response, but the penalty is instability, and 3) a lag in alpha-adrenergic feedforward control improves the stability of the coronary response.