Enhanced caveolae-mediated endocytosis by diagnostic ultrasound in vitro

The modulation of cellular endothelial permeability is a desirable goal for targeted delivery of labels and therapeutic macromolecules; the underlying mechanisms, however, remains poorly understood. Here, we hypothesize that a higher endothelial permeability may result as an outcome of selective enhancement of caveolar endocytosis by ultrasound (US), in the frequency and intensity range of current clinical diagnostic use. To assess the role of free radicals in this phenomenon, we exposed confluent human endothelial cells to pulsed diagnostic US for 30 min, with a mechanical index (MI) of 0.5 and 1.2, using a 1.6-MHz cardiac US scan, and endothelial cells not exposed to US were used as control. Here we show that pulsed diagnostic US with a MI of 1.2 (high mechanical index ultrasound [HMIUS]) were able to selectively enhance endothelial caveolar internalization of recombinant glutathione-S-transferase (GST)-Tat11-EGFP fusion protein (26 1 vs. 11.6 1 A.U, p < 0.001 vs. control), without disruption of plasma membrane integrity. Moreover, pulsed diagnostic US with a MI of 0.5 (low mechanical index ultrasound) did not increase caveolar endocytosis compared with control (11.4 1.2 vs. 11.6 1). Free-radical generation inhibitors, such as catalase and superoxide dismutase, reduced the HMIUS-induced caveolar internalization by a 49.29% factor; finally, HMIUS-induced caveolar endocytosis was found to be associated with a significant increase in the phosphorylation of tyr-14-caveolin1, ser1177-eNOS and Thr202/ Tyr204-ERK? compared with control. These findings show how HMIUS irradiation of human endothelial cells cause a selective enhancement of caveolar-dependent permeability, partially mediated by free radicals generation, inducing a marked increase of phosphorylation of caveolar-related proteins. Thus, the use of diagnostic US could potentially be used as an adjuvant to drive caveolar traffic of extracellular peptides by using a higher level of US energy

Cell-to-cell variability in troponin I phosphorylation in a porcine model of pacing-induced heart failure

We tested the hypothesis that myocardial contractile protein phosphorylation and the Ca2+ sensitivity of force production are dysregulated in a porcine model of pacing-induced heart failure (HF). The level of protein kinase A (PKA)-dependent cardiac troponin I (TnI) phosphorylation was lower in the myocardium surrounding the pacing electrode (pacing site) of the failing left ventricle (LV) than in the controls. Immunohistochemical assays of the LV pacing site pointed to isolated clusters of cardiomyocytes exhibiting a reduced level of phosphorylated TnI. Flow cytometry on isolated and permeabilized cardiomyocytes revealed a significantly larger cell-to-cell variation in the level of TnI phosphorylation of the LV pacing site than in the opposite region in HF or in either region in the controls: the interquartile range (IQR) on the distribution histogram of relative TnI phosphorylation was wider at the pacing site (IQR = 0.53) than that at the remote site of HF (IQR = 0.42; P = 0.0047) or that of the free wall of the control animals (IQR = 0.36; P = 0.0093). Additionally, the Ca2+ sensitivities of isometric force production were higher and appeared to be more variable in single permeabilized cardiomyocytes from the HF pacing site than in the healthy myocardium. In conclusion, the level of PKA-dependent TnI phosphorylation and the Ca2+ sensitivity of force production exhibited a high cell-to-cell variability at the LV pacing site, possibly explaining the abnormalities of the regional myocardial contractile function in a porcine model of pacing-induced HF

Small-animal SPECT and SPECT/CT: application in cardiovascular research

Preclinical cardiovascular research using noninvasive radionuclide and hybrid imaging systems has been extensively developed in recent years. Single photon emission computed tomography (SPECT) is based on the molecular tracer principle and is an established tool in noninvasive imaging. SPECT uses gamma cameras and collimators to form projection data that are used to estimate (dynamic) 3-D tracer distributions in vivo. Recent developments in multipinhole collimation and advanced image reconstruction have led to sub-millimetre and sub-half-millimetre resolution SPECT in rats and mice, respectively. In this article we review applications of microSPECT in cardiovascular research in which information about the function and pathology of the myocardium, vessels and neurons is obtained. We give examples on how diagnostic tracers, new therapeutic interventions, pre- and postcardiovascular event prognosis, and functional and pathophysiological heart conditions can be explored by microSPECT, using small-animal models of cardiovascular disease

Alterations in vasomotor control of coronary resistance vessels in remodelled myocardium of swine with a recent myocardial infarction

The mechanism underlying the progressive deterioration of left ventricular (LV) dysfunction after myocardial infarction (MI) towards overt heart failure remains incompletely understood, but may involve impairments in coronary blood flow regulation within remodelled myocardium leading to intermittent myocardial ischemia. Blood flow to the remodelled myocardium is hampered as the coronary vasculature does not grow commensurate with the increase in LV mass and because extravascular compression of the coronary vasculature is increased. In addition to these factors, an increase in coronary vasomotor tone, secondary to neurohumoral activation and endothelial dysfunction, could also contribute to the impaired myocardial oxygen supply. Consequently, we explored, in a series of studies, the alterations in regulation of coronary resistance vessel tone in remodelled myocardium of swine with a 2 to 3-week-old MI. These studies indicate that myocardial oxygen balance is perturbed in remodelled myocardium, thereby forcing the myocardium to increase its oxygen extraction. These perturbations do not appear to be the result of blunted β-adrenergic or endothelial NO-mediated coronary vasodilator influences, and are opposed by an increased vasodilator influence through opening of KATP channels. Unexpectedly, we observed that despite increased circulating levels of noradrenaline, angiotensin II and endothelin-1, α-adrenergic tone remained negligible, while the coronary vasoconstrictor influences of endogenous endothelin and angiotensin II were virtually abolished. We conclude that, early after MI, perturbations in myocardial oxygen balance are observed in remodelled myocardium. However, adaptive alterations in coronary resistance vessel control, consisting of increased vasodilator influences in conjunction with blunted vasoconstrictor influences, act to minimize the impairments of myocardial oxygen balance

Overview of ventilator-induced lung injury mechanisms

PURPOSE OF REVIEW: Mechanical ventilation is the main supportive therapy for patients with acute respiratory distress syndrome. As with any therapy, mechanical ventilation has side effects and may induce lung injury. This review will focus on stretch-dependent activation of alveolar epithelial and endothelial cells and polymorphonuclear leukocytes, and apoptosis/necrosis balance. RECENT FINDINGS: The past year has seen important research in the area of mechanotransduction and lung native immunity, suggesting further mechanisms of lung inflammation and injury in ventilator-induced lung injury. Research in the past year has also stressed the importance of inflammatory response by alveolar cells and role of polymorphonuclear leukocytes in stretch-induced lung injury and has suggested a role for apoptosis in the maintenance of the alveolar epithelium. SUMMARY: The proportion of patients receiving protective ventilatory strategies remains modest. If efforts to minimize the iatrogenic consequences of mechanical ventilation are to succeed, there must be a greater understanding of the signal transduction mechanisms and the development of potential pharmacologic targets to modulate the molecular and cellular effects of lung stretch