Long-term intermittent compression increases arteriographic collaterals in a rabbit model of femoral artery occlusion.

OBJECTIVES: To assess the effects of intermittent limb compression on arterial collateral formation in a rabbit-model. DESIGN: Animal study. MATERIAL AND METHODS: New Zealand rabbits (n=11), aged 2-years, weight of at least 4.0 kg, underwent bilateral superficial femoral artery ligation. In ten of these, the experimental leg underwent 60 minutes of daily intermittent compression for a ten week period with 3 sec/90 mmHg pressure inflation and a cycle of 3 times per minute. The contra-lateral limbs were not treated. At the end of the ten-week period, high-resolution angiograms were obtained by barium infusion into the aorta. The angiograms were analyzed in a blinded manner and the number of collateral arteries larger than 100 microns, was counted. Following perfusion-fixation, histological specimens of transverse sections of the compressed semi-membranous muscle were examined. RESULTS: The compressed limbs demonstrated a significantly (8.1+/-.87 vs 6.0+/-.97; p<0.005) greater number of collateral vessels, ranging in size from 100-700 microns, as compared to the control sides. The mean size of collaterals in the compressed limbs was not significantly different (0.33+/-0.17 vs 0.31+/-0.16). Microscopic examination of the collaterals confirmed remodeling by a typical neo-intima consisting of 6-7 cell-layers. CONCLUSIONS: Intermittent limb compression increases the number of angiographical collateral arteries.

Pharmacologic pre-conditioning and controlled reperfusion prevent ischemia-reperfusion injury after 30 minutes of hypoxia/ischemia in porcine hearts.

BACKGROUND: Hearts from non-heart-beating organ donors are not transplanted because of risk of ischemia-reperfusion injury. We tested whether pharmacologic pre-conditioning with adenosine and the Na(+)/H(+) exchanger inhibitor, cariporide, combined with controlled reperfusion, would prevent injury in porcine hearts that had sustained 30 minutes of hypoxia/ischemia in closed-chest animals. METHODS: Hearts from Yorkshire pigs (100 kg) were studied in 3 groups. Group 1 (control) hearts were surgically removed while beating. Group 2 hearts were harvested from animals made hypoxic by discontinuing mechanical ventilation for 30 minutes. Group 3 hearts were hypoxic as in Group 2, but these animals received adenosine (40 mg) and cariporide (400 mg) 10 minutes before stopping ventilation. Cardiac function in all groups was assessed ex vivo in a working heart apparatus in which pressure and flow measurements were made over 3 hours. Controlled reperfusion in Group 3 hearts used leukocyte-depleted blood perfusate containing free radical scavengers. Myocardial injury was assessed on the basis of perfusate creatine phosphokinase activity and histopathologically determined injury score. RESULTS: Groups 1 and 3 hearts could be resuscitated to perform work equivalently during the entire reperfusion period and showed positive responses to increases in pre-load and norepinephrine. Group 2 hearts could not perform work. After 3 hours, Group 2 hearts showed significantly higher creatine phosphokinase and histopathologic injury scores compared to with Groups 1 and 3, which were not significantly different from each other. CONCLUSIONS: Pharmacologic pre-conditioning and controlled reperfusion effectively protect non-beating porcine hearts from injury after 30 minutes of hypoxia/ischemia in situ.

Hematopoiesis/erythropoiesis in myocardial infarcts.

Extramedullary hematopoiesis occurring in the myocardium has previously only been reported in a single case of a neonate with cyanotic congenital heart disease. Herein we report the incidental discovery of extramedullary hematopoiesis or pure erythropoiesis in four failing adult hearts with myocardial infarction. In two cases, extramedullary hematopoiesis or erythropoiesis was identified in cardiectomy specimens removed at orthotopic heart transplantation; in two other cases, erythropoiesis was found in left ventricular tissue removed at the time of implantation of left ventricular assist devices. Myocardial hematopoiesis/erythropoiesis was identified based on characteristic light-microscopic findings in routinely processed tissue and was confirmed by immunhistochemistry using monoclonal antibodies to the erythroid cell marker glycophorin A (positive in all cases), the megakaryocyte marker CD61, and the granulocyte marker neutrophil elastase (the latter two markers positive in one case only). None of the four patients had a myeloproliferative disorder or evidence of a myelophthisic process. No hematopoietic elements were identified in 109 cardiectomy specimens without acute or recent infarcts. Myocardial hematopoiesis or erythropoiesis could represent heretofore-unrecognized manifestations of altered cytokine expression in patients with heart failure due to myocardial infarction.

Apoptosis in chronic rejection of human cardiac allografts.

BACKGROUND: We investigated the role of apoptosis (programed cell death) in the pathogenesis of chronic rejection. METHODS: Epicardial coronary arteries from cardiac allografts with chronic rejection were examined for apoptosis by the TUNEL assay. Double labeling was carried out using anti-CD3, anti-CD68, and anti-von Willenbrand factor (vWF) monoclonal antibodies. Additional immunostaining was carried using anti-Fas, anti-Fas-L, and anti-Bcl-2 monoclonal antibodies. Apoptosis-associated oligonucleosomal DNA degradation was assessed by DNA agarose gel electrophoresis. The transcription level of apoptosis-related caspase genes were determined using microarrays. RESULTS: Apoptotic cells (TUNEL+) were detected within the arterial wall and in perivascular areas. Double labeling demonstrated that apoptotic cells included T cells (CD3+), monocyte/macrophages (CD68+), and vascular endothelial cells (VWF+). Numbers and densities of TUNEL+ cells did not correlate with the degree of arterial stenosis. Apoptosis-associated oligonucleosomal DNA degradation was assessed by agarose gel electrophoresis of DNA, which showed DNA fragments of approximately 180 bp and multimers thereof (DNA laddering gel), which are characteristic for DNA fragmentation in apoptotic cells. Microarray analysis demonstrated that the apoptosis related caspases 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, were all transcribed (caspases 8, 9, and 10 were highly up-regulated). These results are consistent with the involvement of apoptosis in chronic rejection. Immunoreactivity for Fas/Fas-L was present at the sites of apoptotic cells. Immunoreactivity for Bcl-2 was present in areas with very few apoptotic cells. CONCLUSIONS: Apoptotic cells include T cells, monocyte/macrophages, and endothelial cells. Apoptosis, likely through the Fas/Fas-L system, is involved in the pathogenesis of chronic rejection in cardiac allografts.

Expression of very low density lipoprotein receptor in the vascular wall. Analysis of human tissues by in situ hybridization and immunohistochemistry.

The recently cloned very low density lipoprotein (VLDL) receptor binds triglyceride-rich, apolipoprotein-E-containing lipoproteins with high affinity. The observation that VLDL receptor mRNA is abundantly expressed in extracts of tissues such as skeletal muscle and heart, but not liver, has led to the hypothesis that this receptor may facilitate the peripheral uptake of triglyceride-rich lipoproteins. However, little information is available concerning the types of cells that express this receptor in vivo. As expression of the VLDL receptor in the vascular wall might have important implications for the uptake and transport of triglyceride-rich lipoproteins, and perhaps facilitate the development of atherosclerosis in hypertriglyceridemic individuals, we used in situ hybridization and immunohistochemistry to determine whether VLDL receptor mRNA and protein was expressed in human vascular tissue. We observed expression of the receptor by both endothelial and smooth muscle cells within normal arteries and veins, as well as within atherosclerotic plaques. In the latter, the VLDL receptor was also expressed by macrophage-derived foam cells. The widespread distribution of the VLDL receptor in vascular tissue suggests a potentially important role for this receptor in normal and pathophysiological vascular processes.

Popliteal venous aneurysm: report of two cases and review of the world literature.

Two new cases of popliteal venous aneurysm are reported and added to the 22 other cases of popliteal venous aneurysm available for review. Both patients were first seen with acute pulmonary embolism and were treated with thrombolytic therapy followed by anticoagulation. Each had recurrent venous thromboembolism before discovery of the popliteal venous aneurysm. One popliteal venous aneurysm was diagnosed with phlebography and the second with venous duplex imaging, confirmed with phlebography. Both were surgically corrected with tangential aneurysmectomy and lateral venorrhaphy. Twenty-four cases of popliteal venous aneurysm are now available for review. Seventy-one percent (17 of 24) presented with pulmonary embolism, 88% (21 of 24) were saccular, and 96% (23 of 24) were located in the proximal popliteal vein. All but two were diagnosed by ascending phlebography. Three patients received no treatment: in two of these the outcome was not documented and the third had occasional pain. Two patients received anticoagulation without subsequent operative repair and both died of recurrent pulmonary emboli. Operative correction resulted in a 75% patency rate with 21% complications, most of which were related to postoperative anticoagulation. No patient who was operated on had subsequent pulmonary embolism, and there were no operative deaths. We suggest that all patients who have pulmonary embolism have lower-extremity venous duplex imaging. All popliteal venous aneurysms should be surgically repaired, inasmuch as nonoperative therapy results in recurrent thromboembolism and an unacceptably high mortality rate. Tangential aneurysmectomy with lateral venorrhaphy is the recommended procedure.

Effects of subcultivation and culture medium on differentiation of human fetal cardiac myocytes.

Previous work has suggested that subcultivated human fetal heart muscle cell cultures contain immature cardiac muscle cells capable only of limited differentiation after mitogen withdrawal. We studied several human fetal heart cultures (14-15 wk gestation) at several passage levels using immunocytochemistry, autoradiography, and Northern blot analysis. Characteristics in high-mitogen (growth) medium were compared with those after serum withdrawal. Cultured cells from one heart, expanded through 2 passages in growth medium, did not beat; however, 75% of cells did beat after subsequent culture for 24 days in low-serum (differentiation) medium containing insulin. In confluent cultures after 1 passage, there was no detectable difference in the number of cardiac myocytes present in growth medium compared with that 7 days after serum withdrawal. After 4 passages, however, serum withdrawal increased the number of cells expressing immunoreactive sarcomeric myosin heavy chain by 100-fold; expression of immunoreactive sarcomeric actin and alpha-cardiac actin mRNA also increased in the same cultures. Similar results were obtained in cultures kept in differentiation medium for 20 days before passage and expansion in growth medium. Using isopycnic centrifugation, a high-density cell fraction was isolated which contained no immunostained myocytes in growth medium but numerous myocytes after serum withdrawal. Combined immunocytochemistry/autoradiography showed that myocytes synthesize DNA in growth medium and in serum-free medium containing fibroblast growth factor, but not in serum-free medium alone. The results indicate that a) human fetal cardiac muscle cells proliferate in vitro and can maintain a phenotype characteristic of fetal myocytes after multiple subcultivations followed by serum withdrawal; b) after subcultivation in growth medium, some myocytes modulate their phenotype into one in which detectable levels of cardiac contractile proteins are expressed only after mitogen withdrawal, and c) the phenotype attained after serum withdrawal is in part dependent on passage level. Cultured human fetal myocardial cells may provide a useful experimental system for the study of human cardiac muscle cell biology.

Glandular cardiac myxomas. Histologic, immunohistochemical, and ultrastructural evidence of epithelial differentiation.

Histologic, histochemical, immunocytochemical, and ultrastructural features of two cardiac myxomas containing glandular elements are reported. Glandular elements in both cases stained positively with both mucicarmine and periodic acid-Schiff reagent with diastase pretreatment (DPAS). Immunoperoxidase studies demonstrated positivity of the glandular cells for carcinoembryonic antigen (CEA), epithelial membrane antigen (EMA), and keratin. Factor VIII-related antigen (FVIIIAg) was identified only in cells lining vascular spaces. Electron microscopic study of one tumor demonstrated well-formed glands having basement membranes, junctional complexes, and apical secretory granules. These findings indicate the capacity for true epithelial differentiation of cardiac myxomas and have implications both as regards the histologic diagnosis of these tumors and their histogenesis.