Debra Graham

The effects of thrombin on bovine aortic endothelial and smooth muscle cells

Recent evidence suggests that thrombin interacts with various cell types, stimulating cellular proliferation and protein and prostanoid production. To further delineate its role in vascular healing, we have studied the effects of thrombin on proliferation and matrix production by the cells of the vessel wall. The addition of thrombin (1 unit/ml) to cultures of bovine aortic smooth muscle cells resulted in an increase in cell proliferation (p less than 0.01) and number (p less than 0.03), whereas in cultures of bovine aortic endothelial cells thrombin produced a decrease in cell proliferation (p less than 0.01) and number (p less than 0.02). Thrombin also altered matrix composition in cultures of these cells. In both bovine aortic endothelial cells and bovine aortic smooth muscle cell cultures grown in the presence of thrombin, total protein content was significantly increased when compared to controls (p less than 0.03). In bovine aortic endothelial cell cultures the addition of thrombin resulted in a decrease in collagen content (p less than 0.01) and an increase in sulfated glycosaminoglycan content (p less than 0.02). In contrast, in bovine aortic smooth muscle cell cultures thrombin resulted in an increase in collagen content (p less than 0.03), whereas glycosaminoglycan content was unaffected. These findings suggest that thrombin may significantly influence vascular healing and function by altering cell number and matrix composition.

Squamous cell carcinoma of the breast: A clinical approach

AbstractBackground: Squamous cell carcinoma (SCC) of the breast is rare. It remains unclear whether a “pure” form of SCC exists or if all known cases actually represent an extreme form of squamous metaplasia within adenocarcinoma. Due to its rarity and controversy ovr its definition, there are no good data on appropriate management and prognosis. Methods: All cases of breast carcinoma indexed at our institution were reviewed to identify seven cases where squamous metaplasia was a significant component of the pathologic diagnosis. Slides and electron micrographs were reviewed by a single pathologist. Clinical information was obtained from medical records. Results: These cases support the concept of a disease continuum with varying degrees of squamous metaplasia. When tumors identified as “pure” SCC on light microscopy are subjected to ultrastructural analysis, either separate squamous and glandular cells are present or both histologic features are noted to coexist in the same cell. Conclusions: Identification of “pure” cases of SCC appears clinically unimportant. All patients presented with advanced disease, thus necessitating aggressive management regardless of histology. Mastectomy was performed due to large tumor size and adjuvant chemotherapy given rather than hormones because of receptor negativity. The mixed histology should direct future drug choice. The role of radiation remains unclear.

The management of localized abdominal aortic dissections

Aortic dissections limited to the abdominal aorta occur infrequently. We have identified four cases of abdominal aortic dissection and have reviewed these in combination with 43 previously reported cases to identify factors that influence the prognosis and management of this disease. Abdominal aortic dissections are similar to thoracic dissections in their presentation, with acute shearing pain and systolic hypertension occurring commonly. Although the diagnosis may be made by ultrasonography or CT scanning, angiography is the definitive diagnostic study. Factors found to be associated with high mortality include presentation with acute pain (p less than 0.0003), involvement of visceral vessels (p less than 0.02), and rupture (p less than 0.000002). Chronicity appears to be protective (p less than 0.04), although chronic dissections may present acutely. Although prosthetic replacement of the involved aorta is the treatment of choice in most cases, nonoperative management with regular follow-up can be considered in asymptomatic chronic dissections.

Outcome of complex vascular and orthopedic injuries of the lower extremity

Thirty-two patients undergoing limb salvage procedures for complex vascular and orthopedic injuries of the lower extremity were studied in order to identify prognostic indicators for delayed amputation in this select group. A high incidence of nerve (38%), soft tissue (66%), and remote injury (47%) was noted. A comprehensive and integrated approach to vascular, orthopedic, and plastic reconstruction was utilized. Of the 32 patients studied, 1 (3.1%) died as a result of remote injury and sepsis. Amputation was required in 9 patients (28%), while 13 (56%) of the patients with limb salvage showed persistent functional or neurologic deficits. Infection was the most significant factor associated with amputation (p less than 0.0005) and was not avoided by the perioperative use of antibiotics. Delayed amputation resulted in a significant extension of total hospitalization (p less than 0.005). The authors favor an aggressive approach to limb salvage with IIIC injury but recommend early amputation in the presence of significant nerve disruption. An attentive use of tissue debridement, intravenous antibiotics, and early wound coverage is needed to limit infection.

Aortic endothelial and smooth muscle cell co-culture: an in vitro model of the arterial wall.

Interactions between vascular endothelial (EC) and smooth muscle cells (SMC) contribute both to the normal function of the vascular wall and to the pathogenesis of lesions such as atherosclerosis and fibrointimal hyperplasia. However, study of these interactions has been hampered by the difficulty in growing these two cell types in simultaneous culture. Methods using conditioned media, shared media, and bilayer culture have been described, but none is well suited to the study of vascular cell interactions. We report a method for EC-SMC co-culture that preserves bilayer morphology, allows independent study of the cells and their matrices after intervention, remains stable over long periods in culture, and permits study of changes in cell-cell interaction with growth of the cells to confluence. This simple bilayer co-culture system simulates the in vivo situation and may enhance our understanding of EC-SMC interactions.

Competitive inhibition of LDL binding and uptake by HDL in aortic endothelial cells

High-density lipoprotein (HDL) may inhibit the binding and cellular uptake of low-density lipoprotein (LDL) as one means of regulating the delivery of exogenous cholesterol to nonhepatic tissues. This may play an important role in atherogenesis, by altering lipid metabolism in cells of the arterial wall. To verify and better characterize this effect, endothelial cells were harvested from bovine aorta and maintained in tissue culture. Following initial preincubation in lipid-deficient culture media, these cells were incubated for 2 hr at 4 degrees C in media containing 125I-LDL (10 micrograms protein/ml) and varying concentrations of either HDL (0-400 micrograms protein/ml) or comparable amounts of Apoprotein A (Apo A), the major protein component of HDL. Intracellular and trypsin-released counts were assayed separately, as a measurement of cellular uptake and membrane bound LDL, respectively. Results of this study indicated an inhibition of LDL binding and uptake by HDL (P less than 0.005, ANOVA). A similar inhibition was found with Apo A alone (P less than 0.005). When identical studies were performed using 125I-Apoprotein B, the protein component of LDL, and Apo A, the latter was found to inhibit the binding of Apo B to the same extent (P less than 0.0006). These results indicate that HDL does inhibit LDL binding and uptake by bovine aortic endothelial cells and that, because this effect is seen equally with only the protein component of these lipoprotein particles, it is most likely due to competitive binding at the receptor level rather than to stearic hindrance or an alteration of the cell membrane.

Oxygen radicals alter LDL permeability and uptake by an endothelial-smooth muscle cell bilayer

There is evidence to suggest that the deposition of lipid within the arterial wall, which is characteristic of atherosclerosis, involves a break in the integrity of the endothelial barrier. Oxidative modification of low density lipoproteins by reactive oxygen species may enhance this process. In this study, an aortic endothelial (EC) smooth muscle cell (SMC) bilayer was briefly exposed to a free-radical generating system to determine the effect of superoxides on lipid permeability and uptake. 125I-LDL (10 micrograms/ml) was added to the EC medium at various time intervals (3, 24, and 48 hr and 9 days). The amount of lipid reaching the subendothelial space was measured. Measurements of EC and SMC binding and uptake of LDL were also obtained and compared with those of untreated cells. The results demonstrate a significant increase in the permeability of the treated EC layer to LDL (P less than 10(-6)), which was sustained over time. Superoxide exposure led to a limited initial increase in EC binding and uptake of LDL, which later returned to control values. In contrast, SMC uptake of LDL was significantly (P less than 10(-3)) and persistently increased over control values and disproportionately increased over cellular binding. Such results suggest that superoxides can increase LDL permeability of the EC barrier. Because this was not associated with a comparable increase in EC binding and uptake, it is unlikely to be due to changes in receptor-mediated, transcellular transport.(ABSTRACT TRUNCATED AT 250 WORDS)

The Association of Intra-Abdominal Infection and Abdominal Wound Dehiscence

Concurrent infection is a risk factor for abdominal wound dehiscence. We reviewed our experience with fascial dehiscence to determine the incidence and to identify prognostic factors for associated intra-abdominal infection. Over a 7-year period, 107 patients with abdominal wound dehiscence were identified. Seventeen were managed nonoperatively, and 90 underwent exploratory laparotomy, 43 of whom had no intra-abdominal pathology and 47 of whom had intra-abdominal infections. Demographic factors, comorbid diseases, and potential indicators of systemic infection did not distinguish patients with intra-abdominal infection from those without. Patients with an intra-abdominal infection were more likely to have undergone an emergency operation (74% vs 48%; P < 0.02), an operation on the colon (55% vs 25%; P < 0.005), or an operation with a higher wound classification (P < 0.02). Mortality was higher in patients with intra-abdominal infection than in those without (44% vs 20%; P < 0.02). Wound dehiscence after emergent operations, and operations with a higher wound classification, especially those involving the colon, should raise concern for intra-abdominal infection. Thorough abdominal exploration should be performed at the time of dehiscence repair. Before nonoperative management is chosen, intra-abdominal infection should be excluded.

Effect of estradiol on low density lipoprotein uptake by bovine aortic endothelial cells

The means by which estrogen retards atherosclerosis cannot be explained solely by changes in circulating lipoprotein levels. We studied the effects of 17 beta-estradiol on the binding, incorporation, and degradation of low density lipoprotein (LDL) by cultured bovine aortic endothelial cells (BAEC). Estrogen receptors in the cytoplasm and nucleus of BAEC could be demonstrated by immunofluorescent staining. Estradiol was found not to affect surface binding of LDL to BAEC. However, at physiologic concentrations (50 pg/ml), estradiol did enhance LDL uptake by the BAEC (P less than 0.005). This enhancement was present but somewhat reduced at higher concentrations of estrogen (P less than 0.05). Only approximately 10% of incorporated LDL was trichloroacetic acid soluble, indicating a low rate of LDL degradation. The relative rate of LDL breakdown within the BAEC was not altered by estrogen. These results, showing estrogen stimulation of LDL uptake by the BAEC, do not clarify the protective effect of this hormone. It is speculated that estrogen may augment the cellular clearance of LDL.

The effect of thrombin on low-density lipoprotein permeability and uptake by an arterial endothelial smooth muscle cell bilayer

Thrombin, a mediator of thrombosis, has been shown to directly alter the function of vascular cells. We studied the effect of thrombin on low-density lipoprotein permeability and uptake by an arterial endothelial cell-smooth muscle cell bilayer to determine its potential role in atherogenesis. Confluent cell bilayers were incubated in media containing thrombin (10 or 50 units/ml) for a period of 24 hours to 9 days. Iodine 125 (125I)-LDL (10 micrograms protein/ml) was then added to the media, and after a 3-hour incubation, 125I-LDL transit through the endothelial cell layer as well as membrane binding and uptake by each cell type were measured. The lower concentration of thrombin caused a delayed increase in both the permeability (p less than 0.0001) and uptake (p less than 0.05) of LDL, but had no effect on membrane binding of the lipoprotein. The higher thrombin concentration led to an immediate increase in endothelial cell permeability to LDL (p less than 10(-7)) and a significant reduction in both cellular uptake (p less than 10(-7)) and membrane binding (p less than 0.0005). In contrast, smooth muscle cell binding and uptake were unaffected at the lower concentration of thrombin. At the higher concentration, smooth muscle cell uptake of LDL was increased (p less than 10(-7)) disproportionately to a more limited increase in membrane binding (p less than 0.05). Endothelial DNA content, reflecting cell number, was increased at 10 units/ml thrombin (p less than 0.001) but markedly reduced at 50 units/ml thrombin (p less than 0.0005), whereas smooth muscle cell DNA content remained unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)