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Biomedical subjects

Rolando E Rumbaut

Publications and source records attributed to Rolando E Rumbaut.

8 recordsLinked to original sources
PubMed2005

Hyperhomocystinemia impairs endothelial function and eNOS activity via PKC activation.

OBJECTIVE: A risk factor for cardiovascular disease, hyperhomocystinemia (HHcy), is associated with endothelial dysfunction. In this study, we examined the mechanistic role of HHcy in endothelial dysfunction. METHODS AND RESULTS: Through the use of 2 functional models, aortic rings and intravital video microscopy of the cremaster, we found that arterial relaxation in response to the endothelium-dependent vessel relaxant, acetylcholine or the nitric oxide synthase (NOS) activator (A23187), was significantly impaired in cystathionine beta-synthase null (CBS(-/-)) mice. However, the vascular smooth muscle cell (VSMC) response to the nitric oxide (NO) donor (SNAP) was preserved in CBS(-/-) mice. In addition, superoxide dismutase and catalase failed to restore endothelium-dependent vasodilatation. Endothelial nitric oxide synthase (eNOS) activity was significantly reduced in mouse aortic endothelial cells (MAECs) of CBS(-/-) mice, as well as in Hcy-treated mouse and human aortic endothelial cells (HAECs). Hcy-mediated eNOS inhibition--which was not rescued by adenoviral transduction of superoxide dismutase and glutathione peroxidase, or by tetrahydrobiopterin, sepiapterin, and arginine supplementations in MAEC--was associated with decreased protein expression and increased threonine 495 phosphorylation of eNOS in HAECs. Ultimately, a protein kinase C (PKC) inhibitor, GF109203X (GFX), reversed Hcy-mediated eNOS inactivation and threonine 495 phosphorylation in HAECs. CONCLUSIONS: These data suggest that HHcy impairs endothelial function and eNOS activity, primarily through PKC activation.

Animals↗
PubMed2005

Neutrophils, nitric oxide, and microvascular permeability in severe sepsis.

STUDY OBJECTIVES: Alterations in microvascular permeability are prevalent in patients with sepsis; a recent study reported that patients with septic shock had increased capillary filtration coefficient (Kf), a noninvasive index of microvascular permeability. We aimed to determine whether patients with severe sepsis had increased Kf, and whether the magnitude of Kf correlated with indexes of nitric oxide activity and neutrophil activation. DESIGN: Single-center, prospective study. SETTING: Twenty-five-bed ICU of a medical college-affiliated teaching hospital. PATIENTS: Fifteen ICU patients with severe sepsis based on the American College of Chest Physicians/Society of Critical Care Medicine consensus criteria of 1992, and 10 nonseptic ICU patients as control subjects. INTERVENTIONS: Kf was measured by venous congestion plethysmography, plasma nitrate/nitrite (NOx) by chemiluminescence, and neutrophil expression of alpha4-integrin (an index of neutrophil activation) by flow cytometry. MEASUREMENTS AND RESULTS: Septic patients had higher Kf than nonseptic control subjects. Kf of septic patients was 5.6 +/- 0.6 x 10(-3) mL.min(-1).100 mL tissue(-1).mm Hg(-1) (mean +/- SEM, mL.min(-1).100 mL tissue(-1).mm Hg(-1) = Kf units [KfU]) as compared to 3.9 +/- 0.5 x 10(-3) KfU in nonseptic ICU patients (p < 0.05). There was no correlation between plasma NOx and Kf, or between neutrophil alpha4-integrin expression and Kf in patients with sepsis. Septic patients with clinical evidence of edema had significantly higher Kf (p < 0.05) than nonedematous septic patients. CONCLUSIONS: ICU patients with severe sepsis have increased Kf, a noninvasive index of microvascular water permeability. The magnitude of hyperpermeability did not correlate with NOx levels or one index of neutrophil activation (alpha4-integrin expression). Presence of peripheral edema in these patients was associated with increased Kf, and may represent a simple, clinical indicator of altered microvascular permeability in sepsis.

Adult↗
PubMed2003

Fluorescent dyes modify properties of proteins used in microvascular research.

OBJECTIVE: Fluorescent dyes, used frequently to label proteins for microvascular experiments, are assumed to not alter the protein's physicochemical characteristics. We tested the validity of that assumption for two probes, bovine serum albumin (BSA) and alpha-lactalbumin. METHODS: Standard electrophoretic techniques were used to examine the influence of five fluorescent dyes on the following three properties of BSA: molecular size (sodium dodecyl sulfate-polyacrylamide gel electrophoresis [PAGE]); relative molecular charge (native PAGE); and isoelectric point (pI) (i.e., isoelectric focusing). Also examined were the influence of the dyes on the relative charge of alpha-lactalbumin (native PAGE). Paired measures of single-vessel flux were made using two commercial preparations of dye-BSA on porcine coronary arterioles and venules. RESULTS: The addition of fluorescein isothiocyanate (FITC) to BSA altered significantly its size (p < 0.01; n = 6), relative charge (p < 0.02; n = 7), and pI. The other dyes caused minimal, yet significant, changes on the relative charge of BSA without influencing size or pI. Only FITC altered the relative charge of alpha-lactalbumin. While arteriolar fluxes of FITC-BSA and tetramethyl rhodamine isothiocyanate (TRITC)-BSA did not differ (p = 0.43; n = 5), in venules the FITC-BSA flux was greater than the TRITC-BSA flux (p < 0.001; n = 6). CONCLUSIONS: The addition of fluorescent dyes, particularly FITC, alters the physicochemical characteristics of two widely used protein probes. These findings may affect the interpretation of microvascular permeability experiments performed using fluorescent dyes, and they suggest care in the selection of dyes for such experiments.

Animals↗
PubMed2002

Similar permeability responses to nitric oxide synthase inhibitors of venules from three animal species.

The influence of nitric oxide (NO) on microvascular permeability remains unclear. NO synthase (NOS) inhibitors have been reported to increase as well as to decrease permeability in different experimental models and animal species. We tested the hypothesis that NOS inhibitors influence venular permeability differently in amphibians and mammals. Permeability coefficients to albumin (P(alb)(s)) were measured on in situ mesenteric venules of the frog and rat and excised pig coronary venules before and after exposure to NOS inhibitors. Despite individual variability in magnitude of responses, NOS inhibitors resulted in a reduction in P(alb)(s) in each species. Superfusion with 10(-5) M N(G)-monomethyl-l-arginine (l-NMMA) reduced P(alb)(s) of frog mesenteric venules by 42% (from a median of 11.4 x 10(-7) cm s(-1), n = 12, P < 0.01) and by 67% in porcine coronary venules (from 12.5 x 10(-7) cm s(-1), n = 5, P < 0.05). The response was attenuated in rat mesenteric venules; 10(-4) M N(G)-nitro-l-arginine methyl ester (l-NAME) reduced P(alb)(s) by 23% (from 7.6 x 10(-7) cm s(-1), n = 9, P = 0.01). The inactive d-enantiomers of the NOS inhibitors were without effect on P(alb)(s) in each model. In pig venules, perfusion with blood modified the permeability responses to l-NMMA, suggesting that effects of NO on permeability are modified by one or more elements of blood. These data support a role of nitric oxide release on venular permeability to albumin that is conserved among the three animal species.

Albumins↗
PubMed2002

Extravascular transport of fluorescently labeled albumins in the rat mesentery.

OBJECTIVE: Fluorescently labeled albumin is used frequently as a tracer when monitoring microvascular permeability. Several fluorescent dyes are available for labeling protein, including fluorescein isothiocyanate (FITC) and Texas Red (TR). Because differences in leakage of dye-labeled proteins have been reported, the objective of the present study was to compare the accumulation of these two tracers in interstitium and lymph after the inflammatory event of ischemia-reperfusion. METHODS: Anesthetized rats were injected intravenously with FITC-labeled albumin (FITC-alb) and TR-labeled albumin (TR-alb) before 30 minutes of mesenteric ischemia. Because the tracers leaked out of the microcirculation after reperfusion, accumulation in the surrounding buffer-superfused tissue, and in separate experiments, accumulation in lymph vessels, was defined as the ratio of tissue-to-plasma and lymph-to-plasma fluorescence. RESULTS: Reperfusion induced a significant increase in tissue-to-plasma fluorescence of FITC-alb; however, no increase was observed for TR-alb. In contrast, lymph-to-plasma fluorescence of TR-alb tended to be greater than FITC-alb. Reperfusion-induced increases in tissue-to-plasma fluorescence of TR-alb occurred only when the superfusate was replaced with mineral oil, in which case tissue-to-plasma TR-alb fluorescence tended to be greater than FITC-alb. CONCLUSIONS: Measurement of fluorescently labeled albumin leakage from mesenteric venules depends on the dye used to label the albumin and requires an assessment of losses from the extravascular measuring region.

Albumins↗
PubMedDate not supplied

Mouse cremaster venules are predisposed to light/dye-induced thrombosis independent of wall shear rate, CD18, ICAM-1, or P-selectin.

OBJECTIVE: Microvascular adhesion of platelets to endothelium occurs in response to various inflammatory stimuli, and in venules is often accompanied by adherent leukocytes. In a light/dye injury model, platelet adhesion and thrombi occur preferentially in venules, though the reasons for this predisposition are unknown. The authors sought to determine whether lower wall shear rates or leukocyte-endothelial interactions accounted for preferential platelet thrombi formation in venules relative to arterioles. METHODS: A light/dye injury model of microvascular thrombosis was used in the mouse cremaster microcirculation. RESULTS: In wild-type mice (n = 17), the time to form microvascular platelet aggregates was delayed in arterioles by 3.1-fold relative to venules (p <.0001). However, arterioles with spontaneously low wall shear rates, as well as arterioles manipulated to reduce wall shear rate to venous levels, still had delayed thrombosis as compared to venules. Similarly, in animals deficient in CD18, P-selectin, or ICAM-1, the time to form platelet thrombi in arterioles was >3.0-fold higher than in venules. CONCLUSIONS: Mouse cremaster venules are predisposed to light/dye-induced microvascular thrombosis. The data suggest that functional differences between arteriolar and venular endothelial cells (independent of wall shear rate and of CD18, P-selectin, and ICAM-1) account for the venular predisposition to thrombosis.

Animals↗
PubMedDate not supplied

Microvascular thrombosis models in venules and arterioles in vivo.

Platelets are intimately involved in hemostasis and thrombosis. Under physiological conditions, circulating platelets do not interact with microvascular walls. However, in response to microvascular injury, platelet adhesion and subsequent thrombus formation may be observed in venules and arterioles in vivo. Numerous intravital video microscopy techniques have been described to induce and monitor the formation of microvascular thrombi. The mechanisms of microvascular injury vary widely among different models. Some models induce platelet activation with minimal effects on endothelium, others induce endothelial inflammation or injury, while other models lead to thrombus formation associated with endothelial denudation. The molecular mechanisms mediating platelet-vessel wall adhesive interactions differ among various models. In some instances, differences in responses between venules and arterioles are described that cannot be explained solely by hemodynamic factors. Several models for induction of microvascular thrombosis in vivo are outlined in this review, with a focus on the mechanisms of injury and thrombus formation, as well as on differences in responses between venules and arterioles. Recognizing these characteristics should help investigators select an appropriate model for studying microvascular thrombosis in vivo.

Animals↗