Expression of Id1 results in apoptosis of cardiac myocytes through a redox-dependent mechanism.

We have constructed a recombinant adenovirus (Ad.Id1) that allows for efficient expression of the helix-loop-helix protein Id1. After infection with Ad.Id1, neonatal cardiac myocytes display a significant reduction in viability, which was proportional to the level of Id1 expression. A similar effect was observed in adult myocytes. Morphological and biochemical assays demonstrated that Id1 expression resulted in myocyte apoptosis. In contrast, expression of Id1 in endothelial cells, vascular smooth muscle cells, or fibroblasts did not affect the viability of these cells. Along with the induction of apoptosis, the expression of Id1 in neonatal cardiac myocytes resulted in an increase in the level of intracellular reactive oxygen species. The source of these reactive oxygen species appears to be the mitochondria. Reducing the ambient oxygen concentration or treatment with a cell-permeant H2O2 scavenger prevented Id1-stimulated apoptosis in cardiac myocytes. These results suggest that the expression of Id1 leads to the induction of apoptosis in cardiac myocytes through a redox-dependent mechanism.

A requirement for the rac1 GTPase in the signal transduction pathway leading to cardiac myocyte hypertrophy.

We have used adenoviral-mediated gene transfer of a constitutively active (V12rac1) and dominant negative (N17rac1) isoform of rac1 to assess the role of this small GTPase in cardiac myocyte hypertrophy. Expression of V12rac1 in neonatal cardiac myocytes results in sarcomeric reorganization and an increase in cell size that is indistinguishable from ligand-stimulated hypertrophy. In addition, V12rac1 expression leads to an increase in atrial natriuretic peptide secretion. In contrast, expression of N17rac1, but not a truncated form of Raf-1, attenuated the morphological hypertrophy associated with phenylephrine stimulation. Consistent with the observed effects on morphology, expression of V12rac1 resulted in an increase in new protein synthesis, while N17rac1 expression inhibited phenylephrine-induced leucine incorporation. These results suggest rac1 is an essential element of the signaling pathway leading to cardiac myocyte hypertrophy.

Protection from reoxygenation injury by inhibition of rac1.

We demonstrate that adenoviral-mediated gene transfer of a dominant negative rac1 gene product (N17rac1) inhibits the intracellular burst of reactive oxygen species (ROS) that occurs after reoxygenation of vascular smooth muscle cells. In contrast, expression of a dominant negative ras gene (N17ras) had no effect. Challenge of control cells and cells expressing N17rac1 with a direct oxidant stress produced an equivalent increase in intracellular ROS levels and subsequent cell death. This suggests that N17rac1 expression appears to block production of harmful oxygen radicals and does not act directly or indirectly to scavenge ROS generated during reoxygenation. Expression of N17rac1 results in protection from hypoxia/reoxygenation-induced cell death in a variety of cell types including vascular smooth muscle cells, fibroblasts, endothelial cells, and ventricular myocytes. These results suggest that reoxygenation injury requires the activation of rac proteins, and that inhibition of rac-dependent pathways may be a useful strategy for the prevention of reperfusion injury in ischemic tissues.

A randomized trial of vascular hemostasis techniques to reduce femoral vascular complications after coronary intervention.

This report details a prospectively randomized clinical trial comparing mechanical clamp compression to hand applied pressure for attaining vascular hemostasis after coronary intervention. Effectiveness was determined by comparing the incidence of femoral vascular complications resulting from each of the 2 techniques. Eligible participants included 778 consecutive patients scheduled for percutaneous coronary intervention over an 8-month period. An unselected cohort of the eligible patients (n = 592), determined by the availability of cross-trained clinicians, underwent follow-up serial physical examinations by blinded observers for the duration of their hospital stay. A second, similarly determined cohort (n = 390), underwent color-duplex ultrasonography within 24 hours of sheath removal. Baseline demographic and clinical characteristics, sheath removal parameters, and subsequent outcomes were collected prospectively. The primary end point was a composite of ultrasound-defined femoral vascular complications: femoral artery thrombosis, echogenic hematoma, pseudoaneurysm, or arteriovenous fistulae formation. Complications diagnosed by physical examination constituted the second fundamental end point and included: persistent oozing, ecchymosis, hematoma, bruit, and pulsatile mass. Compared to manual compression, mechanical clamp hemostasis reduced the primary adverse end point by 63% (p = 0.041). Physical examination detected ecchymosis, oozing, and hematomas at equally high frequencies in the two cohorts. Although 65% of the patients in both treatment groups encountered at least one of these cosmetic complications, the diagnoses made by physical examination did not correlate with ultrasound-defined pathology. Multivariable stepwise logistic regression analysis identified a relationship of advanced age and lower body weight to vascular complications. Utilization of a mechanical clamp rather than conventional hand pressure to attain vascular hemostasis significantly reduces ultrasound-defined femoral vascular pathology. Discrepancies between physical examination and ultrasound diagnoses challenge the utility of clinical assessment alone and establish ultrasound as the diagnostic modality of choice.

Effect of a dominant negative ras on myocardial hypertrophy by using adenoviral-mediated gene transfer.

BACKGROUND: The small guanosine triphosphate-binding protein ras regulates a signal transduction cascade linking cell surface receptors to mitogen-activated protein kinase (MAPK). Because the molecular signaling mechanisms underlying cardiac hypertrophy remain unclear, the current study examined the regulatory role of ras in both the biochemical and morphologic aspects of hypertrophy. METHODS: Adenoviral-mediated gene transfer was used to express a dominant negative mutant of ras (rasN17) at high efficiency in primary neonatal ventricular myocytes. Beta-galactosidase staining and Western blot analysis confirmed successful transfection and expression of the rasN17 gene product. MAPK activity was measured by an in vitro kinase assay resulting in radioactive phosphorus labeled product. Morphologic hypertrophy was assessed by fluorescein-conjugated phalloidin. RESULTS: Compared with uninfected or control adenoviral-infected cells, myocytes infected with rasN17 demonstrated attenuated basal MAPK activity. In contrast, rasN17 expression did not affect endothelin 1-induced MAPK activation. Morphologic studies showed that although rasN17 produced a phenotypic difference in the basal state, the ability of cardiac myocytes to morphologically respond to endothelin 1 stimulation, as manifested by sarcomeric reorganization, remained unaltered by the expression of the rasN17 gene product. CONCLUSIONS: Endothelin 1-stimulated MAPK activation and endothelin 1-induced morphologic hypertrophy are ras-independent processes.

Rac1 is required for cell proliferation and G2/M progression.

We have transiently expressed a dominant negative form of rac1 (N17rac1) using adenoviral-mediated gene transfer. The level of N17rac1 expression is demonstrated to be proportional to the multiplicity of infection. Expression of N17rac1 in Rat 2 fibroblasts results in cytostatic growth arrest. Cell-cycle analysis demonstrates that cells expressing N17rac1 accumulate in G2/M. These results suggest that rac1 is required for cell proliferation and provide the first demonstration in mammalian cells of a role for small GTP-binding proteins in the G2/M transition.

Composition of lung lavage in pulmonary alveolar microlithiasis.

A case of pulmonary alveolar microlithiasis (PAM) is reported wherein total lung lavage was performed for relief of dyspnea. Characterization of the lavage material and examination of the microliths isolated from the lavage fluid confirmed previous reports of their spherical-ovoid shape and a 2:1 calcium to phosphate composition. The microliths contained considerable amounts of ionizable iron and generated oxidants in an in vitro system. A detailed biochemical analysis of the lavage fluid reflected elevations in total protein, phosphatidylserine, phosphatidylglycerol and the ratio of phosphatidylglycerol to phosphatidylinositol. Surfactant apoprotein-A levels approximated that of normal patients. The potential roles of oxidant generation and alterations in surfactant metabolism are discussed in the context of the pathogenesis of PAM.

Brain computerized tomography after hyperbaric oxygen therapy for carbon monoxide poisoning.

The role of brain computerized tomography (CT) imaging in predicting clinical outcome was investigated in patients receiving hyperbaric oxygen therapy for serious carbon monoxide (CO) poisoning. From a series of 48 consecutive patients suffering loss of consciousness from CO exposures, the records of 40 selected patients were evaluated to determine how their CT findings correlated with clinical outcome. A neuroradiologist blinded to patient outcome confirmed the radiographic findings. CT abnormalities consisted of globus pallidus hypodensities (nine patients), subcortical white matter hypodensities (four), cerebral cortical lesions (one), cerebral edema (one), hippocampal lesions (one), and complete loss of gray-white differentiation (one). Of the patients with globus pallidus lesions, 44% manifested incomplete recovery, whereas white matter lesions reflected a 74% incidence of morbidity. Age, duration of CO exposure, and interval between CO exposure and treatment did not significantly relate to clinical outcome. The blood carboxyhemoglobin levels correlated with clinical prognosis (P < 0.05) and, importantly, CT results significantly predicted clinical outcome (P < 0.05). A normal scan correlated highly with a complete recovery, whereas an abnormal scan predicted incomplete recovery or death, despite prior HBO therapy. The current study establishes prognostic validity for brain CT imaging for evaluating clinical outcome after HBO therapy for CO poisoning.

The development of spontaneous colo-umbilical fistula.

A patient with colo-umbilical fistula is reported. This presentation is unique because it documents the development of a fistula from a colonic diverticulum. Sigmoid colectomy was undertaken successfully.

Pituitary-thyroid axis reactivity to hyper- and hypothyroidism in the perinatal period: ontogeny of regulation of regulation and long-term programming of responses.

To evaluate the role of perinatal thyroid status in the development of pituitary-thyroid axis regulation, we administered triiodothyronine to newborn rats for the first five days postpartum to achieve hyperthyroidism, or propylthiouracil perinatally to rat dams and pups from gestational day 17 through postnatal day 5 to achieve hypothyroidism. Plasma T4, T3, and TSH levels were determined from birth through 50 days postpartum. Administration of exogenous T3 produced the expected immediate suppression of plasma T4 and TSH, with recovery toward normal values beginning within days of discontinuing the T3 regimen. Plasma T3 values were markedly elevated during the period in which T3 was being given, but subsequently became subnormal, with deficits persisting into young adulthood. With the PTU regimen, plasma T4 and T3 levels were markedly suppressed through postnatal day 10, rose over the ensuing two weeks, but nevertheless showed significant deficits into adulthood. TSH levels in the immediate neonatal period were subnormal in the PTU group, despite the marked lowering of circulating thyroid hormones; TSH then rose dramatically to levels four times normal, subsiding to control values by the end of the first month. These results suggest that a critical period exists in which regulation of pituitary-thyroid axis function is programmed. During this phase, TSH secretion can be suppressed by excess thyroid hormones, but cannot be increased by hormone deficiencies. Perhaps more importantly, perinatal thyroid status "programs" its own future reactivity, so that early hypothyroidism results in reduced T4 and T3 levels in adulthood, despite normal levels of TSH.

Thyroid hormone regulates ontogeny of beta adrenergic receptors and adenylate cyclase in rat heart and kidney: effects of propylthiouracil-induced perinatal hypothyroidism.

In mature animals, thyroid hormone is permissive for beta adrenergic receptor expression and adrenergic control of adenylate cyclase. To determine if endogenous thyroid hormones play a similar role in the development of receptors and transduction mechanisms, we administered propylthiouracil perinatally to rat dams and pups from gestational day 17 through postnatal day 5. Circulating thyroid hormones were completely suppressed through postnatal day 10 and then rose to only slightly subnormal values by the 3rd to 4th postnatal week. In the heart, hypothyroidism completely suppressed the initial development of beta adrenergic receptor binding sites, with recovery paralleling the return of thyroid hormone levels. In contrast, development of basal and isoproterenol-stimulated adenylate cyclase activity showed more lasting deficiencies with a delayed onset corresponding to general growth impairment; however, forskolin-stimulated adenylate cyclase developed in a nearly normal pattern. Effects on development of renal beta receptors and adenylate cyclase were of smaller magnitude and comprised only the delayed onset phase; receptor deficiencies appeared after 10 days and adverse effects on adenylate cyclase were limited to the isoproterenol-sensitive component, consisting of a shift of the ontogenetic peak to later ages. Endogenous thyroid hormones thus contribute two distinct factors to beta receptor/adenylate cyclase development: they are obligatory for cardiac beta receptor development, but also, in parallel with general effects on growth and development, serve to program the ontogeny of transduction factors linking the receptors to adenylate cyclase. The predominance of propylthiouracil effects on isoproterenol-stimulated adenylate cyclase but not on enzymatic responses to forskolin suggests that thyroid hormones may be controlling the development of regulatory G-proteins.

Thyroid hormone differentially regulates development of beta-adrenergic receptors, adenylate cyclase and ornithine decarboxylase in rat heart and kidney.

In mature animals, thyroid hormone produces parallel up-regulation of beta-adrenergic receptor binding sites and their linkage to adenylate cyclase; during development, these same processes may be critical in establishing the set-point for subsequent adrenergic reactivity. In the current study, we administered triiodothyronine to neonatal rats for the first five days postpartum and evaluated [125I]pindolol binding capabilities and adenylate cyclase activity in membrane preparations from heart and kidney. In the heart, hyperthyroidism elicited an initial increase in receptor density, with subsequent deficits and an eventual return to normal values by young adulthood. In contrast, the ability of isoproterenol, a beta-adrenergic agonist, to stimulate adenylate cyclase was enhanced regardless of whether receptor numbers were increased or decreased; the same effects were also present for basal adenylate cyclase activity and non-receptor-mediated stimulation by forskolin. Enhanced cyclase activity involved both increases in the magnitude of response as well as accelerated onset of the postweaning peak of enzyme activity, results which suggest a direct impact of thyroid status on the ontogenetic expression of adenylate cyclase itself. The kidney, which possesses less efficient beta-receptor coupling to adenylate cyclase in the neonate, was less drastically affected by triiodothyronine for either beta-receptor binding sites or enzyme activity. As we had previously shown that neonatal hyperthyroidism uncouples beta-receptors from growth-related enzymes, such as ornithine decarboxylase, we also evaluated whether the promotion of adenylate cyclase responses was mechanistically linked to effect on ornithine decarboxylase; administration of cyclic AMP analogs to 5 days-old rats led to inhibition of the enzyme in the heart, whereas the same treatment in 9 days-old animals was ineffective. These data suggest that thyroid hormone differentially regulates the development of beta-receptors as well as adenylate cyclase and ornithine decarboxylase, with preferential effects on tissues, such as the heart, that already possess efficient linkage of the receptors to cell transduction mechanisms at birth.

Role of thyroid hormone in the development of beta adrenergic control of ornithine decarboxylase in rat heart and kidney.

The role of thyroid status in the ontogeny of beta adrenergic receptor control of ornithine decarboxylase (ODC) activity was assessed in hearts and kidneys of neonatal rats. Hyperthyroidism induced by administration of tri-iodothyronine on postnatal days 1 to 5 caused a reduction in the ability of isoproterenol to stimulate cardiac ODC but subsequently accelerated the onset of the postweaning peak of the response; the latter effect was even more prominent when tri-iodothyronine administration was given on postnatal days 14 to 18. Hypothyroidism induced by propylthiouracil administration led to persistent subsensitivity of the cardiac ODC response to beta receptor stimulation. Kidney ODC, which does not become subject to beta receptor regulation until after weaning, was resistant to hyperthyroid-induced changes in reactivity, but hypothyroidism still resulted in long-term response deficits. These results suggest that thyroid hormone is permissive for normal development of the beta receptor-ODC link, and that the euthyroid state provides the optimal conditions for maturation of this signal transduction mechanism. The relative resistance of kidney ODC responses to alterations by hyperthyroidism further indicates that the effects of excess hormone can only be expressed when the receptor-enzyme link is already competent. Finally, thyroid status had equivalent effects on the abilities of vasopressin or angiotensin to stimulate ODC, suggesting that the site of thyroid hormone action is at a transduction locus common to several different receptor types.

Moyamoya disease associated with polycystic kidney disease and eosinophilic granuloma.

Moyamoya disease has been associated with renal artery stenosis, cerebral hemorrhage, and multiple cranial traumas. We report a unique case of moyamoya disease associated with polycystic kidney disease and eosinophilic granuloma. Although the etiology of moyamoya disease is unknown, a familial pattern of occurrence has been documented. Of particular importance is its presentation with polycystic kidney disease, an autosomal dominant disease, suggesting a hereditary component to the etiology of this unusual vasculitic disease.