A model of BAX gene delivery to human lung cancer.

In this report we have investigated the effects of BAX in enhancing apoptosis in two primary non-small cell lung cancer cell lines. A count of the apoptotic cells by TUNEL staining revealed that almost 70% of BAX over-expressing cells died, while very few apoptotic cells were detectable in the wildtype cells or in the cells transfected with an empty vector. These findings suggest that de-regulated expression of BAX may provide a novel mechanism for initiating cell death in non-small cell lung cancer cells. Further studies are needed to better define the involvement of this protein in the complex mechanism of lung carcinogenesis and to definitely demonstrate the therapeutic utility of targeting this pathway.

The effect of different isolation techniques on human osteoblast-like cell growth.

Surgical treatment of post-traumatic or neoplastic bone defects often represents a problem in orthopaedic routine. Autologous tissue always stands for the first choice material. The recent therapeutic approaches for tissue repair of bone defects attempt to mimic the natural process of bone repair by delivering a source of cells capable of differentiating into osteoblasts. In this study two different types of human osteoblast cell harvesting were compared in primary cell culture to evaluate the best way to obtain cells for clinical use. Numerous articles describe the characteristics of each one of these systems, but there is no report comparing the influence of these different isolation techniques on cell growth. Cultures from 22 bone specimens obtained from donors were established in two different ways and their proliferation was compared. An enzymatic procedure to extract human osteoblasts (hOBcol) was used in one group; spontaneous cells outgrowth, human osteoblasts (hOBsog) was expected in the other group. Cells of both groups were characterised as osteoblasts by immunohistochemical staining with Bone Morphogenetic Protein-2,4 (BMP-2,4), expression of collagen type I as well as the amount of alkaline phosphatase activity (ALP) detected in the cell cultures. We found that the time needed in primary cultures till confluence was dependent on the age of the donors as well as on the method of cell harvesting. Cells from under 65-year old donors were growing significantly faster by the hOBcol method as compared to hOBsog 20.57 +/- 2.99 days vs. 30.00 +/- 4.36. Cells harvested from donors older than 65 years of age needed 23.88 +/- 2.95 in the hOBcol compared to 34.25 +/- 4.27 days in the other group. In the experimental cultures, after one passage with trypsin/EDTA, there was a significant difference between the two groups. There was an improved cell growth in the hOBsog group found on days 8, 9 and 10 of cultivation. Immunohistochemical staining as well as ALP activity were similar in both groups. In conclusion this study evaluated an important step for a tissue engineering approach to the repair of bone defects, which may have clinical applications in post-traumatic orthopaedic surgery.

Preservation of the pulmonary valve complex in tetralogy of fallot: how small is too small?

BACKGROUND: Surgical repair of congenital lesions associated with right ventricular outflow tract obstruction frequently requires the destruction of pulmonary valve (PV) components including the valve annulus. The resultant pulmonary insufficiency may lead to late functional deterioration of right ventricular performance. Acute right ventricular dysfunction has been associated with poor pulmonary runoff, tricuspid valve regurgitation, and pulmonary hypertension. Preservation of PV competence may prevent both early and late right ventricular failure. However, the recent trend towards earlier repair of tetralogy of Fallot (TOF) may preclude preservation of the PV in favor of a transannular patch. We reviewed our experience with surgical repair of TOF to determine if age and/or body size affected the ability to repair the PV. METHODS: We reviewed the clinical records of 50 consecutive children who underwent surgical repair of TOF by one surgeon. The latter 27 patients underwent repair with an intention to preserve their pulmonary valve. In total, 28 patients underwent repair with preservation of their pulmonary valve, and form the basis of this study. Serial echocardiographic assessments were performed early (3 to 6 months) and late (12 months) after surgery. RESULTS: Pulmonary valve preservation was possible in the majority of children (89%) in whom it was intended. Pulmonary valve competence was observed in 68% of children, with only 5 (16%) children demonstrating severe insufficiency at follow-up. Residual right ventricular outflow tract obstruction was present in only 1 child who underwent repair with pulmonary valve preservation at greater than 2 years of age. CONCLUSIONS: Our data suggest that earlier repair of TOF does not preclude preservation of the pulmonary valve and may indeed facilitate repair. The pulmonary valve remains competent at 12 months, with acceptable gradients, and should participate in somatic growth. Pulmonary valve preservation during repair of TOF may prevent free pulmonary insufficiency, progressive right ventricular dilation, and the need for future prosthetic pulmonary valve replacement.

Cardiac rhabdomyomas and obstructive left heart disease: histologically but not functionally benign.

BACKGROUND: Cardiac rhabdomyoma is the most common primary heart tumor in infants. Spontaneous regression of such tumors is common, particularly with smaller lesions, followed by resolution of symptoms. Based on our data on spontaneous involution, our institutional philosophy has been one of expectant management in the absence of life-threatening symptoms. However, surgical intervention sometimes is required for the extirpation of a rhabdomyoma from the left ventricular outflow tract. METHODS: A retrospective review was conducted of 30 children in whom a rhabdomyoma was diagnosed over a 27-year period. RESULTS: Twenty-three percent (7/30) of the children required surgical extirpation of the tumor from within their left ventricular outflow tract, although a total of 94% had left ventricular involvement. There were no deaths. To date, no child has required reexcision of tumor. CONCLUSIONS: The natural history of rhabdomyoma is one of spontaneous regression (the 23 children who did not undergo surgical intervention are alive and continue to be followed up medically). We recommend surgical excision to alleviate acute outflow tract obstruction with reliance on the tumor's natural history of regression to achieve long-term freedom from reoperation. Although operation has been recognized as lifesaving, we were somewhat surprised to find that greater than 20% of our pediatric population required operative intervention.

Relentless pulmonary vein stenosis after repair of total anomalous pulmonary venous drainage.

BACKGROUND: Progressive stenosis of the pulmonary veins after repair of total anomalous pulmonary venous drainage is frequently refractory to surgical therapy. METHODS: Retrospective review of 170 consecutive patients treated for total anomalous pulmonary venous drainage identified 13 patients with postrepair pulmonary vein stenosis. Preoperative and operative data were analyzed to define the patterns of progression and efficacy of surgical techniques. RESULTS: Seventeen reoperations were performed in 13 patients. Postrepair pulmonary vein stenosis was most common in the infracardiac and mixed subtypes (p < 0.02). All 4 patients with unilateral stenosis, 2 of whom had progression of stenosis resulting in nearly complete unilateral pulmonary vein occlusion, survived. Six of 9 patients with bilateral disease died (p < 0.05 versus unilateral); 2 of the 3 survivors were repaired with a novel technique creating a sutureless neoatrium without evidence of restenosis at 1.8 years' follow-up. Stenting was uniformly unsuccessful. CONCLUSIONS: In unilateral stenosis, progression of disease may be survivable with loss of single-lung perfusion. Although bilateral disease is lethal in most cases, creation of a sutureless neoatrium has demonstrated short-term freedom from disease progression.

Surgical management of total anomalous pulmonary venous drainage: impact of coexisting cardiac anomalies.

BACKGROUND: Recent reports have cited improving results for surgical management of isolated total anomalous pulmonary venous drainage. Complex cases (with other cardiac anomalies) are less frequently reported and are associated with higher mortality. METHODS: Retrospective review identified 170 consecutive patients treated for total anomalous pulmonary venous drainage from 1982 to 1996: 44 cases were "complex" (with significant associated cardiac lesions) and 126 cases were "simple." RESULTS: Operative mortality for simple cases decreased from 26% to 8%, and mortality for complex cases remained constant at 52%. Age, size, and the presence of atrial isomerism were univariate predictors of mortality. Multivariable analysis identified only univentricular hearts and associated cardiac lesions as predictors of operative mortality. Pulmonary artery (n = 16) and arteriopulmonary (n = 7) shunting strategies for complex cases resulted in less than 30% long-term survival. CONCLUSIONS: Despite improvement in survival for simple cases, management of total anomalous pulmonary venous drainage with single-ventricle hearts or other associated cardiac lesions remains problematic.

Contractile function of human veins after long-term storage in different media.

We wished to determine the preservation of contractile function of human saphenous veins during long-term storage (36 and 96 h) at low temperatures in different solutions. Two crystalloid solutions (Krebs-Henseleit and Bretschneider's cardioplegic solution), as well as heparinized blood and albumin solution as used in cardiac surgery were compared. Contractile function of human saphenous vein ring segments was tested in a vessel myograph. Potassium chloride (KCl 80 mM) was used to achieve receptor-independent maximal contraction; contractility to norepinephrine (NE) was tested in a concentration-dependent manner (10(-8)-10(-6)M). Nitroglycerin (NTG) was used to test vascular relaxation. Thirty-six hours of storage in 5% human albumin abolished responses to KCl and to NE. At this time, contraction of vein ring segments stored in heparinized blood was decreased to 24% in response to KCl and to 16% in response to NE. After 36-h storage in Bretschneider's cardioplegic solution, contractile function was not significantly decreased. The contraction was still 82% after KCl and was unchanged after NE. After 96 h, the contractile response was markedly impaired in all vein ring segments, and no relevant differences were evident between the solutions. In all experiments, NTG caused complete relaxation. Because Bretschneider's cardioplegic solution preserved vascular contractile function for 36 h, the implementation of this solution might enable long-term storage of human vessels for transplantation, bypass grafts, and in vitro experiments.

Reactivity to alpha agonists is heightened in immature porcine pulmonary arteries.

BACKGROUND: Pulmonary hypertension after cardiopulmonary bypass is a common problem in pediatric cardiac operations. This study tested the hypothesis that there is a difference between adult and immature pulmonary artery constrictor and dilator responses. METHODS: Reactivity of pulmonary artery ring segments from 22 mature (15 to 19 weeks) and 15 immature pigs (4 to 5 weeks) was tested in a vessel myograph. Potassium as a receptor-independent vasoconstrictor and phenylephrine as an alpha-receptor-mediated vasoconstrictor were used to assess smooth-muscle vasoconstriction. To assess endothelial cell function (nitric oxide production and secretion), we used increasing concentrations of bradykinin or acetylcholine. Sodium nitroprusside was used to produce maximum smooth-muscle relaxation at the end of each experiment. RESULTS: The data demonstrated maturation-independent endothelium and smooth-muscle-mediated vasodilation. Pulmonary artery ring segments from immature pigs had significantly less KCl constriction compared with mature pigs (p < 0.05). In contrast, pulmonary ring segments from immature pigs demonstrated enhanced alpha-receptor-mediated constriction compared with mature pigs. CONCLUSIONS: These data may explain perioperative pulmonary vasoconstriction in pediatric patients.

Pulmonary artery endothelial cell function in swine pseudomonas sepsis.

A substantial increase in pulmonary vascular resistance is associated with sepsis and its sequelae (sepsis syndrome and septic shock). It is postulated that increased resistance may result from sepsis-induced endothelial cell injury or altered vasoreactivity secondary to pulmonary hypertension. We, therefore, tested the hypothesis that sepsis causes endothelial cell injury and that increased pulmonary pressure alters vascular reactivity. Young swine (15-25 kg) were anesthetized and ventilated. Septic animals received a 1-hr infusion of live Pseudomonas aeruginosa (n = 11), and the control cohort received 0.9% NaCl (n = 7). All animals were studied for 300 min following the infusion. Postmortem branches of peripheral pulmonary arteries were prepared and tested in a vessel myograph. Ring segments were set to 90% of the circumference the vessels would have at pressures of 20, 30, 40, or 50 mmHg (L90), corresponding to varying pulmonary pressures observed in sepsis. A high dose of potassium was used to obtain maximum possible contraction. Prostaglandin was used to precontract the vessels before testing endothelial cell responses to acetylcholine or bradykinin. Sodium nitroprusside was added at the end of each experiment to obtain maximum possible smooth muscle relaxation. No differences in contraction or relaxation were observed when vessels were set to different pressures (i.e., 20 vs 50 mmHg). Maximum possible contraction to KCl was significantly decreased after 300 min of sepsis compared to control. No differences between groups were found in contractility to prostaglandin. Bradykinin-induced EDRF/NO production, mediated by BK2 receptors, was not altered in Pseudomonas sepsis (97-98% of total relaxation control and 91-95% septic cohort). Response to acetylcholine was significantly decreased after sepsis (89-95% of total relaxation control and 51-61% of septic cohort relaxation). Decreased response to acetylcholine could not be attributed to decreased smooth muscle sensitivity to nitric oxide because the response to bradykinin plus sodium nitroprusside was not altered following sepsis. Vessel reactivity was not altered by increasing pressure settings reflective of changing pulmonary pressure in vivo. These results strongly suggest a sepsis-induced alteration in pulmonary artery endothelial cell receptor sensitivity to acetylcholine, independent of changing pulmonary arterial pressures. This is the first time this decrease has been shown in pseudomonas sepsis.

Microvascular dysfunction after myocardial ischemia.

Endothelium-mediated relaxation and smooth muscle function in large coronary arteries are resistant to prolonged global ischemia. We used a small-vessel myograph to test the hypothesis that small intramyocardial artery endothelium and smooth muscle function have greater sensitivity to ischemic injury than large artery endothelium and smooth muscle. Normothermic global ischemia was induced in 15 porcine hearts. Intramyocardial arterial ring segments were assessed at 0, 30, 60, 90, and 120 minutes of ischemia in vitro with a small-vessel myograph. Potassium determined smooth muscle contraction, bradykinin endothelium-mediated relaxation, and sodium nitroprusside direct smooth muscle relaxation. Endothelium-mediated relaxation after 30 minutes of ischemia was similar to control (56% versus 66%) but was impaired at 60, 90, and 120 minutes of ischemia (32%, 11%, and 6%). Smooth muscle contraction was unchanged at 30 and 60 minutes compared with control (56 and 53 versus 63 mm Hg) but was significantly decreased at 90 and 120 minutes (33 and 13 mm Hg). Direct smooth muscle relaxation was significantly decreased at 120 minutes of ischemia compared with control (58% versus 95%). In a previous study, epicardial coronary artery endothelium-mediated smooth muscle vasodilation and direct smooth muscle vasodilation were preserved until 160 minutes of ischemia. After 160 minutes of ischemia, endothelium-mediated relaxation was lost and only direct smooth muscle vasodilation was preserved. In contrast to vasodilation, vasoconstriction was significantly reduced at 140 minutes of ischemia. These data indicate a greater and earlier adverse effect of ischemia on intramyocardial arterial endothelium-mediated relaxation than smooth muscle contraction or relaxation. These data support the hypothesis that there is an early functional endothelial cell injury associated with global ischemia. Relaxation that is endothelium-dependent in intramyocardial arteries is more sensitive to ischemic injury than in epicardial arteries. Unique to this study was the evaluation of small intramyocardial arteries (281 +/- 29 microns) that are the primary sites of coronary vascular resistance. Microvascular endothelial dysfunction after ischemia, therefore, may contribute to the "no-reflow phenomenon" seen during reperfusion injury.

Ischemia and activated neutrophils alter coronary microvascular but not epicardial coronary artery reactivity.

Activated neutrophils have been implicated in reperfusion injury and the no-reflow phenomenon of intramyocardial arterioles. This study tested the hypothesis that ischemia and activated neutrophils impair coronary endothelial and smooth muscle cell function of epicardial and intramyocardial coronary arteries. Alteration of smooth muscle and endothelial cell function in epicardial coronary arteries (3 mm diameter) and intramyocardial coronary arteries (0.3 mm diameter) was compared by means of a myograph after exposure to ischemia (epicardial, 160 minutes, intramyocardial, 30 minutes), activated neutrophils, and combined ischemia and activated neutrophils. Morphologic studies at the ultrastructural level were done by means of scanning electron microscopy. Epicardial coronary artery function was normal after ischemia, storage with activated neutrophils, and ischemia followed by storage with activated neutrophils. Intramyocardial artery function, however, was altered. Contraction to a 45 mmol/L concentration of potassium chloride after ischemia and storage with activated neutrophils was increased (p = 0.06). Smooth muscle relaxation was significantly decreased after ischemia, but storage with activated neutrophils did not further decrease smooth muscle relaxation. Endothelium-dependent relaxation to bradykinin was significantly decreased after combined ischemia and incubation with activated neutrophils (p < 0.05). Sensitivity to bradykinin was decreased after both ischemia alone (p < 0.05) and activated neutrophils alone (p < 0.05). Similar morphologic alterations were found in epicardial and intramyocardial arteries after ischemia. Activated neutrophils alone minimally damaged endothelial cells of nonischemic intramyocardial and epicardial arteries. Endothelial cells of both arteries exposed to ischemia alone showed evidence of ischemic damage, including endothelial cell blebbing, nuclear bulging, and appearance of large holes in the cell surface. Severe endothelial cell damage was found after combined ischemia and storage with neutrophils: total destruction of cells and exposure of the basal lamina. Endothelial damage, therefore, correlated with artery function in intramyocardial but not in epicardial arteries. These results indicate that ischemia is a prerequisite for severe neutrophil injury of intramyocardial artery endothelium-mediated relaxation. This may explain no-reflow phenomenon in arterioles concurrent with myocardial reperfusion injury.

Changes following in vitro endothelial cell lining of ePTFE prostheses: late morphologic evaluation of six failed grafts.

This paper describes the morphologic appearance during long term follow-up of in vitro endothelialised ePTFE grafts (IVECL) implanted in patients with crural reocclusions. Between June 1989 and December 1990, 13 femorocrural IVECL bypasses were implanted. Follow-up angiograms demonstrated stenoses in the middle of the graft in six patients. Two of these patients developed symptoms, and the grafts were biopsied approximately 1.5 years after implantation during a patchplasty procedure. The remaining four patients with asymptomatic stenoses refused elective reoperation and suffered a graft occlusion 53 to 619 days after implantation, all leading to amputation. Biopsy specimens and explanted grafts were examined with standard and electron microscopy. Both biopsies demonstrated multiple layers of degenerating myofibroblasts (MFB). The four explanted grafts also showed altered MFB in addition to necrosis of the graft surface. No endothelial cells were seen on any of the preparations. Long term follow up of IVECL protheses in the crural position has demonstrated that it is possible to lastingly bind cells on an artificial surface. Whether the MFB found are a substitute of lost endothelial cells, or are an end product of metaplastic and/or degenerative alterations, can only be clarified through further biopsy studies.

Effect of triiodothyronine on postischemic myocardial function in the isolated heart.

Thyroid dysfunction has been shown to have a significant impact on hemodynamic status and cardiac function. The purpose of this study was to determine the influence of triiodothyronine (T3) on cardiac functional recovery after ischemia in a dose-dependent manner. Postischemic functional recovery was assessed in isolated rabbit hearts mounted in a modified Langendorff preparation. Left ventricular systolic, diastolic, and peak developed pressures were measured before and after ischemia, and calculated as a percentage of preischemic function. Two cohorts of hearts were studied: the first was exposed to warm ischemia until a myocardial contracture of 4 mmHg was produced; the second cohort was exposed to warm ischemia until a contracture of 15 mm Hg was observed. In each cohort, T3 was added to the perfusion solution after ischemia in a physiologic concentration (2.5 x 10(-9) g/mL; 1 x T3), as well as ten times (2.5 x 10(-8) g/mL; 10 x T3) and a hundred times (2.5 x 10(-7) g/mL; 100 x T3) the physiologic concentration. One group, given the carrier only but without T3, served as the control. Rabbit hearts exposed to a short period of ischemia (4-mmHg diastolic contracture) showed increased recovery with 1 x T3 and 10 x T3. 100 x T3 did not bring about improved left ventricular recovery versus that in the control group. Rabbit hearts in the 15 mm Hg-diastolic contracture cohort showed increased recovery with 10 x T3 but not with 1 x T3. 100 x T3 led to decreased recovery in this cohort versus that in the control group.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of triiodothyronine on stunned myocardium.

Administration of thyroid hormone, triiodothyronine (T3), causes numerous cardiovascular effects such as increases in stroke volume, cardiac output, heart rate, and myocardial contractility, and decreases in systemic vascular resistance. Along with other stressors, cardiopulmonary bypass (CPB) has been associated with reduced levels of T3. We examined the effects of T3 on early postischemic myocardial recovery in rabbit hearts subjected to crystalloid perfusion to simulate a low T3 state, and in pig hearts following global ischemia due to CPB. Studies using the former system showed that T3 administration results in significantly improved developed pressure after reperfusion of mildly ischemic hearts compared to controls, without producing inotropic effects. In more severely stunned rabbit hearts, physiologic and 10 times physiologic doses of T3 produced significantly improved (p < 0.05) stroke work end-diastolic length compared to placebo treatment. T3 treated pigs undergoing CPB and subjected to 30 minutes of global normothermic ischemia experienced significantly enhanced recovery of left ventricular contractility compared to controls at 90 and 120 minutes post reperfusion. Neither placebo nor T3 affected myocardial adenosine triphosphate levels. These data show that T3 enhances recovery from myocardial stunning without producing acute inotropic effects.

Implantation of in vitro endothelialized polytetrafluoroethylene grafts in human beings. A preliminary report.

To assess the impact of in vitro endothelialization on prosthetic graft patency, we performed femorotibial reconstruction in four patients. Polytetrafluoroethylene grafts (6 mm), lined with cultivated autologous endothelial cells, harvested from the veins of the forearm, were used. Autologous endothelial cells were harvested enzymatically and characterized by morphology and factor VII staining. After a cultivation period of 17 to 23 days, the cell count increased from 27 +/- 3 x 10(4) endothelial cells to 5.4 +/- 1.1 x 10(6). Endothelial cell seeding on polytetrafluoroethylene prostheses was then performed. To improve endothelial cell attachment to the graft surface, polytetrafluoroethylene grafts (60 to 70 cm; 6 mm diameter) were precoated with fibrin glue containing fibrin and fibronectin and the fibrinolysis inhibitor aprotinin. Seeding density of 49 +/- 10 x 10(3) endothelial cells per square centimeter yielded a preconfluent monolayer immediately after seeding, as demonstrated by scanning electron microscopy. A second cultivation period of 6 days, after seeding and before implantation, was necessary for establishment of a confluent monolayer and to allow for maturation of the endothelial cell cytoskeleton as well as production and excretion of extracellular matrix. Grafts endothelialized in vitro were implanted in four patients requiring femorotibial reconstruction. Scintigraphic studies with indium 111-labeled platelets demonstrated little or no platelet deposition, indicating persistent endothelialization. All grafts remained patent at 3 months after implantation.

New aspects of the degeneration of bioprosthetic heart valves after long-term implantation.

Bioprosthetic heart valves removed 76 to 150 months after implantation were morphologically investigated to correlate structural alterations with clinical failure modes. Traditional morphologic methods of evaluating valvular heterografts, such as microradiography and electron microscopy, were complemented by undecalcified ground sections, a new technique for analyzing the distribution of mineral deposits. Apart from well-investigated mechanisms that accelerate tissue degeneration, our observations point to additional facts: (1) phagocytosis of collagen fibrils and elastic material by macrophages and foreign body giant cells in areas near tears and perforations and (2) initial calcification indicated by delicate crystals in the intercellular space arranged in close relation to the periodicity of the cross-striation pattern of collagen fibrils. The present report not only calls attention to degenerative changes that are enhanced by mechanical stress but also underlines phagocytosis as an important mechanism in the destruction of bioprosthetic heart valves.

Prospective clinical study with in vitro endothelial cell lining of expanded polytetrafluoroethylene grafts in crural repeat reconstruction.

A nonrandomized prospective clinical study was undertaken to evaluate the technique and efficacy of in vitro endothelial cell lining of synthetic grafts. Twenty-six patients (10 men and 16 women with a mean age of 68.4 years; range, 49 to 80 years) with end stage chronic peripheral vascular disease requiring reoperation were entered into the study. In 13 patients venous endothelial cells were harvested 4 to 7 weeks before operation, grown to confluency in culture flasks, and seeded onto the inner surface of expanded polytetrafluoroethylene grafts. Thirteen patients received untreated expanded polytetrafluoroethylene grafts and served as a control. A scoring system with use of intraarterial angiography was used to assess disease severity. No statistically significant differences in angiographic score were seen between the two groups, indicating comparable severity of disease. Early secondary graft patency (0 to 30 days) was 92% for the in vitro endothelial cell lining group and 53% for control patients. The amputation rate after 18 months for the in vitro endothelial cell lining group was 15%, with a 31% rate in the control group. The functional performance of the in vitro endothelial cell lining bypasses was superior to that of untreated bypass grafts during the observed follow-up period. These early results suggest that in vitro endothelial cell lining is a method that can reduce the early occlusion rate now seen after repeat reconstruction of crural vessels.