John T. Fleming

Hyperhomocysteinemic Diabetic Cardiomyopathy: Oxidative Stress, Remodeling, and Endothelial-Myocyte Uncoupling

Accumulation of oxidized-matrix (fibrosis) between the endothelium (the endothelial cells embedded among the myocytes) and cardiomyocytes is a hallmark of diabetes mellitus and causes diastolic impairment. In diabetes mellitus, elevated levels of homocysteine activate matrix metalloproteinase and disconnect the endothelium from myocytes. Extracellular matrix functionally links the endothelium to the cardiomyocyte and is important for their synchronization. However, in diabetes mellitus, a disconnection is caused by activated metalloproteinase, with subsequent accumulation of oxidized matrix between the endothelium and myocyte. This contributes to endothelial-myocyte uncoupling and leads to impaired diastolic relaxation of the heart in diabetes mellitus. Elevated levels of homocysteine in diabetes are attributed to impaired homocysteine metabolism by glucose and insulin and decreased renal clearance. Homocysteine induces oxidative stress and is inversely related to the expression of peroxisome proliferators activated receptor (PPAR). Several lines of evidence suggest that ablation of the matrix metalloproteinase (MMP-9) gene ameliorates the endothelial-myocyte uncoupling in diabetes mellitus. Homocysteine competes for, and decreases the PPARγ activity. In diabetes mellitus, endothelial-myocyte uncoupling is associated with matrix metalloproteinase activation and decreased PPARγ activity. The purpose of this review is to discuss the role of endothelial-myocyte uncoupling in diabetes mellitus and increased levels of homocysteine, causing activation of latent metalloproteinases, decreased levels of thioredoxin and peroxiredoxin, and cardiac tissue inhibitor of metalloproteinase (CIMP) in response to antagonizing PPARγ.

Two week nicotine treatment selectively increases bone vascular constriction in response to norepinephrine

This study was designed to determine if nicotine treatment alters the constrictor and/or dilator function of the vessels which regulate blood flow to intact bone. Nicotine (1.7 mg/kg/day) or nicotine‐free, phosphate‐buffered saline was administered subcutaneously to mature male rats for 2 weeks via osmotic mini‐pumps. On the 14th day, the rats were anesthetized and in vivo experiments were performed to quantitate the changes in arterial blood pressure and perfusion of the intact tibia (measured by laser Doppler flowmetry) in response to two constrictor agonists (norepinephrine, NE and arginine vasopressin, AVP) and two vasodilator agents (acetylcholine, ACh and sodium nitroprusside, SNP). Dose‐response curves were generated by plotting the change in the bone vascular resistance index (mmHg/bone perfusion units) evoked by each dose of agonist. In addition, bone arteriolar expression of endothelial nitric oxide synthase protein was quantitated by Western blot analysis. Nicotine treatment significantly enhanced the constriction of the bone vasculature in response to NE, but not to AVP. Vascular dilation in response to ACh and SNP was not changed by nicotine. These results indicate that nicotine selectively accentuates the constrictor response of the bone vasculature to exogenous NE. This enhanced constriction to NE is not due to impaired endothelial cell release of nitric oxide or diminished smooth muscle response to nitric oxide. Since NE and AVP activate similar cell signaling mechanisms to induce constriction, the selective enhancement of NE‐induced constriction suggests that nicotine alters a mechanism unique to NE signaling; possibly the number or binding affinity of alpha adrenergic receptors. Since endogenous NE regulates basal blood flow to bone, the effect of nicotine to augment NE‐induced constriction could lead to a chronic reduction in blood flow to bone.

Bone Blood Flow and Vascular Reactivity

Blood flow is essential for normal bone growth and bone repair. Like other organs, the regulation of blood flow to bone is complex and involves numerous physiologic mechanisms including the sympathetic nervous system, circulating hormones, and local metabolic factors. Our studies addressed the following questions: (1) Which endogenous vasoconstrictor agents regulate in vivo blood flow to bone? (2) Does a decrease in bone vascular reactivity to vasoconstrictor hormones account for the increase in blood flow during bone healing? (3) Does the endothelium influence bone arteriolar function? An intact bone model was developed in the rat to assess hormonal regulation of in vivo bone blood flow and in vivo bone vascular reactivity. An isolated, perfused bone arteriole preparation was employed to characterize the responsiveness of small resistance-size arterioles (diameter < 100 µm) to vasoconstrictor hormones and to evaluate the role of the vascular endothelium to modulate vascular smooth muscle reactivity. Our results indicate that: (1) though exogenous endothelin is a potent constrictor of the in vivo bone vasculature, endogenous endothelin does not actively regulate in vivo blood flow; (2) the increase in blood flow to a bone injury site is not due to a decrease in bone vascular sensitivity to norepinephrine, and (3) isolated bone arterioles of young rats are very sensitive to vasoconstrictor hormones but exhibit only modest endothelium-mediated vasodilation.

Hyperhomocysteinemia decreases bone blood flow

Elevated plasma levels of homocysteine (Hcy), known as hyperhomocysteinemia (HHcy), are associated with osteoporosis. A decrease in bone blood flow is a potential cause of compromised bone mechanical properties. Therefore, we hypothesized that HHcy decreases bone blood flow and biomechanical properties. To test this hypothesis, male Sprague–Dawley rats were treated with Hcy (0.67 g/L) in drinking water for 8 weeks. Age-matched rats served as controls. At the end of the treatment period, the rats were anesthetized. Blood samples were collected from experimental or control rats. Biochemical turnover markers (body weight, Hcy, vitamin B12, and folate) were measured. Systolic blood pressure was measured from the right carotid artery. Tibia blood flow was measured by laser Doppler flow probe. The results indicated that Hcy levels were significantly higher in the Hcy-treated group than in control rats, whereas vitamin B12 levels were lower in the Hcy-treated group compared with control rats. There was no significant difference in folate concentration and blood pressure in Hcy-treated versus control rats. The tibial blood flow index of the control group was significantly higher (0.78 ± 0.09 flow unit) compared with the Hcy-treated group (0.51 ± 0.09). The tibial mass was 1.1 ± 0.1 g in the control group and 0.9 ± 0.1 in the Hcy-treated group. The tibia bone density was unchanged in Hcy-treated rats. These results suggest that Hcy causes a reduction in bone blood flow, which contributes to compromised bone biomechanical properties.

Reduced Renal Microvascular Reactivity to Angiotensin II in Diabetic Rats

Objective: Renal hyperfiltration in early diabetes is often correlated with increased renal blood flow, reflecting dilation of resistance arterioles. This loss of arteriolar tone has been associated with an impaired reactivity to angiotensin II (Ang II). This study determined if outer cortical arterioles (preglomerular and/or postglomerular) of diabetic rats exhibit a diminished reactivity to Ang II and established if renal vascular prostaglandins account for the diminished responsiveness.

Endogenous norepinephrine regulates blood flow to the intact rat tibia

The goal of our study was to determine if endogenous norepinephrine (NE) has a role in the regulation of basal blood flow to intact bone. The experimental plan was to measure bone blood flow before and after pharmacological blockade of alpha‐adrenergic receptors. A significant increase in blood flow after receptor blockade would suggest that endogenous norepinephrine exerts a tonic constrictor effect on the vessels supplying blood to the bone. Mature, male rats were anesthetized with Inactin. Arterial blood pressure and left tibia blood flow (laser Doppler flowmetry) were measured. A cannula was inserted into the right iliac artery and advanced to the aortic bifurcation to deliver drugs into the left hindlimb circulation, including the left tibia vasculature. Bolus injection of norepinephrine caused a dose‐dependent decrease in bone blood flow (30–40%). Blockade of alpha‐adrenergic receptors with phentolamine or phenoxybenzamine attenuated by more than 50% the norepinephrine‐induced decrease in bone blood flow. In separate rats that had not received exogenous norepinephrine, injection of phentolamine alone decreased bone vascular resistance by 34 ± 3%. Similarly, phenoxybenzamine decreased resistance by 25 ± 4%. These results are consistent with the conclusion that alpha‐adrenergic receptors mediate a significant constriction of blood vessels which participate in the partial control of basal blood flow to the intact rat tibia.

Tyrosine kinase receptor alteration of renal vasoconstriction in rats is sex- and age-related

Male rat renal blood vessels undergo reduced contraction to norepinephrine with aging. There is a greater renal vascular impairment in male compared with female rats. We investigated specific tyrosine kinase receptor inhibition of renal interlobar artery responsiveness to phenylephrine in male and female rats at specifically designated ages. Vessels from young male rats contracted much less to phenylephrine when the vessels were pretreated with the tyrosine kinase inhibitors Lavendustin A, HNMPA-(AM)₃, or AG1478. Vessels from adult female rats pretreated with Lavendustin A showed no difference in contraction from control, but did demonstrate a slightly reduced contraction when pretreated with AG1478. Middle-aged male rat vessels treated with Lavendustin A demonstrated no inhibition, but the insulin and epidermal growth factor receptor (EGFR) antagonists both induced a decline in contraction. Vessels from aged male rats demonstrated no effect related to the 3 pretreatments. Middle-aged and aged female rats pretreated with any inhibitor demonstrated no inhibitor-dependent alterations. We conclude that maximum contraction of interlobar arteries from adult male rats is reduced when tyrosine kinase receptor activity is reduced. Female rats demonstrated much less inhibitor-related change of contraction.

Kidney Blood Flow Changes in Shock

The syndrome of multiple-system organ failure (MSOF) was originally suggested by Baue [1] to be the sequential failure of two or more organ systems in critically ill patients. These initial patients were ill from diverse causes such as pancreatitis, heart disease and a leaking colon anastomosis. This orig inal observation was later confirmed and placed in to better focus by Eiseman [2] and Fry [3] where systemic infection was the identified common ethiologic thread. Although these reports devel oped mostly following multiple trauma, MSOF is currently recognized in a multitude of scenarios such as volume or cardiogenic shock, systemic in fection or the persistent foci of dead tissue. Invasive infection from an identifiable source need not be present for the development of the syndrome, but clearly the circulatory and hypermetabolic state commonly identified in sepsis is a constant feature of MSOF, implying activation of the inflammation cascade as an etiologic factor.

The Influence of Extracellular Calcium on Microvascular Tone in the Rat Cremaster Muscle

Abstract In vivo Responses of arterioles and venules to changes in bath calcium concentrations were observed in the cremaster muscle of male Sprague-Dawley rats. Small arterioles (2A, 3A) initially exposed to a solution containing calcium (2.55 mM) significantly dilated in response to a 0-calcium bath. Reexposure to calcium (>0.65 mM) caused 2A and 3A arterioles to constrict to diameters similar to the initial control values. In contrast, large arterioles (1A) and all venules (1V, 2V, 3V) were unresponsive to exposure to a 0-calcium solution or to reexposure to calcium (0.65–5.10 mM). Treatment with mefenamic acid (10 μg/ml), a prostaglandin synthesis inhibitor, produced marked constriction of arterioles but not of venules, suggesting the involvement of endogenous vasodilator prostaglandins in the regulation of resting diameters of arterioles. In the presence of mefenamic acid, 1A arterioles dilated when exposed to a 0-calcium solution and constricted back to control diameters following reintroduction of calcium into the bath. These data demonstrate heterogeneity in the responsiveness of cremasteric microvessels to changes in extracellular calcium. The small arterioles were most responsive to calcium. The lack of response by the largest arterioles appears to be due to the dilator influences of endogenous prostaglandins.

Dietary Myo-inositol Restores Diabetic Renal Arteriolar Reactivity to Angiotensin II but not to Norepinephrine

Objective: This study addresses the hypothesis that the diminished constriction of renal arterioles to angiotensin II (Ang II) and norepinephrine (NE) in diabetic rats is due to elevated activity in the polyol pathway. This activity results in reduced incorporation of myo‐inositol into membrane phospholipids and impaired signal transduction.