Chemotaxis of aortic endothelial cells in response to fibronectin.

We have determined the chemotactic response of bovine aortic endothelial cells to fibronectin and to endothelial cell mitogens (endothelial cell growth supplement, tumor extract), using blind-well chemotaxis chambers. Fibronectin induced a chemotactic response in bovine aortic endothelial cells; at 100 micrograms/ml, chemotaxis increased by 440% above that observed in negative controls (p less than 0.001). The chemotactic response plateaued with time, paralleling the disappearance of the fibronectin concentration gradient in the chambers. As further evidence that chemotaxis was measured, we observed that cell migration did not occur when cells were incubated with fibronectin in the absence of a concentration gradient. Endothelial cell mitogens increased bovine aortic endothelial cell proliferation in our experiments but did not stimulate chemotaxis above background levels. In contrast, fibronectin inhibited cell proliferation by 23%. We present a hypothetical model of the role of fibronectin in evoking endothelial cell chemotaxis during tumor neovascularization.

Fibronectin-induced protein carboxy-O-methylation in aortic endothelial cells.

In a previous report (Bowersox, J.C. and Sorgente, N. (1982) Cancer Res. 42, 2547-2551) we demonstrated that the glycoprotein fibronectin is a chemoattractant for vascular endothelial cells. In probing the mechanisms by which fibronectin induces endothelial cell chemotaxis, we have discovered that the carboxy-O-methylation of cellular proteins is stimulated by fibronectin. By measuring the incorporation of L-[methyl-3H]methionine into alkali-labile, [3H]methyl ester linkages, we determined that fibronectin stimulated protein carboxy-O-methylation in aortic endothelial cells in a time- and concentration-dependent manner; the greatest stimulation occurred at 100 micrograms/ml fibronectin (approx. 35% above controls). When inhibitors of carboxymethylation were added, fibronectin-induced stimulation of protein methylation did not occur. Furthermore, inhibitors of methylation prevented the chemotaxis of endothelial cells in response to fibronectin. These data support our hypothesis that fibronectin mediates endothelial cell chemotaxis, such as that occurring during neovascularization. As carboxy-O-methylation of cell proteins is also effected by fibronectin, transmethylation reactions may be an important component of endothelial cell chemotaxis.

Altered growth kinetics of dermal fibroblasts and arterial smooth muscle cells from spontaneously diabetic BB rats.

We cultured arterial smooth muscle cells and dermal fibroblasts from spontaneously diabetic BB rats and normoglycemic littermate controls. Although there were no detectable differences in the morphology of cells obtained from diabetic rats, significant differences existed in growth parameters of the diabetic smooth muscle cells. These cells grew more rapidly than smooth muscle cells from normal rats (population doubling times: normal = 42.6 +/- 3.2 h, diabetic = 31.8 +/- 3.7 h) and attained greater densities at confluence. The growth rates of the diabetic smooth muscle cells were dependent on the initial seeding density of cells, a characteristic not observed in cells from normal rats. Although growth rates of the diabetic smooth muscle cells were increased, their plating efficiencies were reduced. Dermal fibroblasts from diabetic rats grew at the same rates as fibroblasts from control animals and the plating efficiencies of the diabetic fibroblasts were increased rather than reduced. In these studies, we have shown that vascular-derived cells from diabetic rats have growth defects not detected in dermal fibroblasts from the same animals. This emphasizes the importance of using blood vessel cells to probe the pathophysiology of diabetic vascular disease. Furthermore, our results establish the validity of using spontaneously diabetic rats as a model system for examining inherent cell defects in insulin-dependent diabetes mellitus.

Efficacy of a fibrin hemostatic bandage in controlling hemorrhage from experimental arterial injuries.

OBJECTIVE: To determine if a pressure dressing containing fibrinogen and thrombin could provide more effective control of arterial hemorrhage than a pressure dressing alone in an animal model of arterial injury. DESIGN: Randomized acute (nonsurvival) experiment in swine. SETTING: Federal biomedical research institute. ANIMALS: Six anesthetized Yorkshire swine. INTERVENTIONS: Uncontrolled arterial hemorrhage was induced in anesthetized swine by creating femoral artery lacerations. Hemorrhage was controlled by a gauze bandage containing fibrinogen and thrombin, applied with 1 minute of 3.5-kg pressure. The dressings were left in place for 1 hour after the pressure was removed. The contralateral limbs received identical treatment with plain gauze dressings. MAIN OUTCOME MEASURES: Total blood loss, mean arterial pressure, and mortality were measured after 1 hour. RESULTS: After 1 hour, blood loss in the fibrin bandage group was 123 +/- 48 mL, compared with 734 +/- 134 mL in the control group (P = .0022). In the group treated with the fibrin bandages, there was no significant decrease in the mean arterial pressure after arterial laceration. In contrast, there was a decrease of 30 mm Hg in the group treated with gauze dressings alone. There was no animal mortality during the study period. CONCLUSIONS: Bandages containing fibrinogen and thrombin significantly reduced the amount of blood loss and allowed mean arterial pressures to be maintained in animals with uncontrolled hemorrhage from femoral artery lacerations. A hemostatic bandage may be an important adjuvant for controlling severe extremity hemorrhage in the prehospital setting.

Remote operative urology using a surgical telemanipulator system: preliminary observations.

OBJECTIVES: A prototype surgical telemanipulator system was evaluated to determine the feasibility of remotely performing operative urology. METHODS: Surgeons operated remotely on anesthetized swine (n = 4), using a four degree-of-freedom teleoperator system. A stereoscopic video display of the remote surgical field guided tissue manipulation, which was performed using standard instrument tips attached to robotic arms. Nephrectomies, cystotomy closures, and ureteral anastomoses were performed by surgeons linked only by electronic cables to the remote operative field (5 m away). Flexible fiberoptic endoscopy was performed remotely, as well, using a simulated ureter in an ex vivo testing apparatus. The endoscope was positioned and manipulated remotely, and was maneuvered through a 25-cm-long tube. RESULTS: All procedures were completed successfully, demonstrating the capability to remotely incise, dissect, suture, and ligate tissues. Operative times were prolonged; however, the surgical telemanipulator was precise and accurate. Surgeons guided a flexible fiberoptic endoscope by remote observation and manipulation through 5-mm-diameter simulated ureters. There were no operative complications. CONCLUSIONS: Telesurgical manipulation is feasible in a controlled, experimental environment. All critical tasks performed by surgeons can be completed remotely, although performance must be improved substantially before telemanipulators enter the clinical armamentarium. Major hurdles remain in identifying clinical needs, improving performance and safety, and developing telecommunications capabilities that will enable telesurgical systems to achieve regulatory approval and acceptance.

Use of an intuitive telemanipulator system for remote trauma surgery: an experimental study.

BACKGROUND: Death from battlefield trauma occurs rapidly. Potentially salvageable casualties generally exsanguinate from truncal hemorrhage before operative intervention is possible. An intuitive telemanipulator system that would allow distant surgeons to remotely treat injured patients could improve the outcome from severe injuries. STUDY DESIGN: We evaluated a prototype, four-degree-of-freedom, telesurgery system that provides a surgeon with a stereoscopic video display of a remote operative field. Using dexterous robotic manipulators, surgical instruments at the remote site can be precisely controlled, enabling operative procedures to be performed remotely. Surgeons (n = 3) used the telesurgery system to perform organ excision, hemorrhage control, suturing, and knot tying on anesthetized swine. The ability to complete tasks, times required, technical quality, and subjective impressions were recorded. RESULTS: Surgeons using the telesurgery system were able to close gastrotomies remotely, although times required were 2.7 times as long as those performed by conventional techniques (451 +/- 83 versus 1,235 +/- 165 seconds, p < 0.002). Cholecystectomies, hemorrhage control from liver lacerations, and enterotomy closures were successfully completed in all attempts. Force feedback and stereoscopic video display were important for achieving intuitive performance with the telesurgery system, although tasks were completed adequately in the absence of these sensory cues. CONCLUSIONS: We demonstrated the feasibility of performing standard surgical procedures remotely, with the operating surgeon linked to the distant field only by electronic cabling. Complex manipulations were possible, although the times required were much longer. The capabilities of the system used would not support resuscitative surgery. Telesurgery is unlikely to play a role in early trauma management, but may be a unique research tool for acquiring basic knowledge of operative surgery.

Microvascular anastomoses performed in rats using a microsurgical telemanipulator.

OBJECTIVE: To determine the feasibility of performing microsurgical procedures with a remote telemanipulator using a rat femoral artery anastomosis model. MATERIALS AND METHODS: A remote telemanipulator system was developed that enabled precision movements to be performed at up to 30x magnification. Ten 1-mm femoral artery anastomoses were performed in rats using the telemanipulator, and results were compared to those from a control group in which the procedure was performed with conventional microsurgical techniques. Study endpoints included anastomosis completion time, short-term patency, and procedural complications. Statistical analysis was performed using Student's t-test. RESULTS: All anastomoses performed by remote telemanipulation and by conventional microsurgery were completed successfully. Anastomosis completion times were 100.0 +/- 18.6 minutes in the telemanipulator group and 38.8 +/- 5.0 minutes using conventional techniques (p < 0.001). Patency in both groups at 5 minutes and at one hour was 100%. No intraoperative complications were encountered. Postmortem ex vivo examination of the excised arterial segment revealed no technical defects in either group. CONCLUSIONS: Complex procedures requiring a high degree of precision and dexterity can be performed using an electromechanical interface specifically designed for micromanipulation. Performance limitations are similar to those previously reported for remote surgical teleoperation, and most likely reflect incompletely characterized restrictions on multi-sensory information.

Data-based resource planning for battlefield trauma management.

Optimal medical care of the wounded soldier can be provided only if clinicians have appropriate supplies present in adequate quantities. At the same time, the battlefield environment requires maximum efficiency in resource utilization. Logistical support for field surgical units can be allocated more efficiently without compromising the quality of care by using a database derived from trauma patient management. The records of patients treated for gunshot wounds at an urban level I trauma center were reviewed and the use of medications at each stage of treatment was quantified. From this information, a database was compiled that could be used to predict medical resource requirements for combat scenarios. Such a database could be used to assist in planning logistical support, resulting in better tailoring of deployable medical unit resources to meet the actual needs of injured soldiers.

New technology in U.S. Army health care: the role of laparoscopic cholecystectomy.

In the near future, laparoscopic cholecystectomy could become one of the most frequently performed surgical procedures in U.S. military hospitals. To determine the best way to implement this new technology in the U.S. Army Health Services Command (HSC), we have assessed the safety, efficacy, and health consequences of laparoscopic cholecystectomy. Approximately 2,800 open cholecystectomies are performed annually in HSC hospitals, 11% of them on active duty personnel. If cholecystectomies were performed using laparoscopic surgical techniques on active duty patients who meet currently accepted indications, 3,360 personnel duty days lost to convalescence and 7,048 days of limited duty (time on physical profiles) would be recouped. Applying the procedure to other beneficiaries (dependents and retired service members) would result in a potential annual savings of 11,000 hospital bed days. If implemented using stringent criteria for training and certification, these savings could be achieved with no increase in patient morbidity or mortality and with significant improvement in patient outcome.

Army trauma research: tapping the potential.

Trauma management is the primary mission of military surgeons. Since the Vietnam War, however, military surgeons have relinquished leadership in clinical trauma care to the civilian sector, particularly to urban university surgeons. In this paper we explore whether the Army's contribution to trauma research has also diminished. Using standard bibliometric analysis of publication counts, we have shown that few recent publications related to trauma have originated from U.S. Army Medical Centers, compared with adjacent civilian universities. In 1988, 16 papers originating from the eight Army Medical Centers had key words related to trauma. In contrast, eight universities adjacent to the Army's medical centers published 139 articles on trauma. Problems including lack of clinical exposure to trauma patients, lack of funding, and inadequate emphasis on staff research training have contributed to this decline. We review these factors and describe solutions that could reverse this trend.

Complex task performance in Cyberspace. Surgical procedures in a telepresence environment.

To assess the capabilities of our fully functional, prototype telepresence surgery system, experienced surgeons performed complete operative procedures on live, anesthetized pigs. Cholecystectomy, the prototypical procedure for evaluating the integration of surgical skills, was successfully performed in six animals. There were no aborted attempts or complications. Other procedures completed included gastrotomy and enterotomy closures, anastomosis of the small intestine, and nephrectomy. No specific training was required for using the telepresence surgery system, and the "feel" of the system was described as intuitive. Operative times were longer than required in conventional, open surgery, most likely the result of the four degrees of freedom available in the manipulators of the current-generation system. Force feedback and high-resolution, stereoscopic video input facilitated performance. Surgeons operating through a first-generation telepresence system can achieve technical results equivalent to those obtained in conventional surgery.

In vivo collagen metabolism in spontaneously diabetic (db/db) mice.

To further define the pathogenesis of diabetic connective tissue lesions, collagen synthesis and degradation were measured in vivo in spontaneously diabetic db/db mice. A double isotopic labeling technique, in which 14C-labeled and 3H-labeled proline were injected into the same mouse 7 days apart, was applied. Collagen synthesis and degradation were assessed in skins, intestines, hearts, and kidneys. There were no changes in collagen metabolism in the intestines of the diabetic mice. In all other tissues, collagen degradation was accelerated. Collagen synthesis was decreased in skins, but increased in the hearts and kidneys of the diabetic mice. These tissue-specific changes in collagen metabolism resulted in a net loss of collagen in all tissues examined except intestines. The results of this study provide insight into the mechanisms leading to connective tissue defects occurring in diabetes mellitus.

Trauma and military applications of blood substitutes.

PURPOSE: To review potential clinical uses of erythrocyte substitutes in treating military battlefield casualties, with specific emphasis on combat injury rates and wounding patterns, resuscitation doctrine and logistic requirements. METHODS: Review of published medical literature and of unclassified documents from the U.S. Armed Forces Blood Program. RESULTS: Hemorrhage is the leading cause of death on the battlefield. Early intervention, with definitive treatment, could save up to 30% of soldiers who are killed in action or who die of wounds. Hemorrhage control and rapid volume expansion in appropriate casualties are the main priorities in pre-hospital resuscitation of battlefield casualties. The role for oxygen-carrying fluids in the initial management of military injuries is undefined; however, erythrocyte substitutes could reduce the logistic requirements for blood in field hospitals. In recent wars, outdating of stored blood resulted in 60-95% of units being discarded: 60% of 1.3 million units in Vietnam and 95% of 120,000 units in the Persian Gulf War. CONCLUSIONS: Safety, long storage life, light unit weight, and tolerance to environmental extremes are all characteristics that are necessary for erythrocyte substitutes to extend or replace the use of stored blood in treating battlefield casualties.

Plasma fibronectin metabolism during hemorrhagic shock and starvation.

In this report we characterize the response of the plasma protein fibronectin to hemorrhagic shock and starvation, conditions associated with decreased function of mononuclear phagocyte system (MPS). Rats were starved for 3 days, then half of the animals were subjected to fixed-volume hemorrhagic shock by removing an estimated 35% of their blood volumes for 20 min. After volume replacement, animals were injected iv with [14C]valine. At time points up to 10 hr after hemorrhage, plasma fibronectin concentrations and fibronectin synthesis were quantitated. In additional rats treated identically, fibronectin clearance was assessed by measuring the disappearance of 125I-fibronectin from the plasma. When compared to control animals, either starvation or hemorrhagic shock produced similar perturbations in plasma fibronectin metabolism; fibronectin concentrations were reduced from 241.3 +/- 34.6 micrograms/ml (mean +/- SEM) (controls) to 123 +/- 32.6 micrograms/ml (starvation) or 150.0 +/- 13.0 micrograms/ml (hemorrhage). Plasma [14C]fibronectin specific radioactivities, indicative of fibronectin synthesis, were also significantly reduced. Hemorrhagic shock in rats that previously had been starved did not depress fibronectin concentrations or synthesis to a greater extent than starvation alone. The rates of 125I-fibronectin clearance were increased in starvation and hemorrhagic shock (t1/2 = 233.0 +/- 13.0 min, controls; 174.6 +/- 10.7 min, starvation; 167.4 +/- 13.6 min, hemorrhage). In contrast to changes observed in fibronectin metabolism, total plasma protein concentrations were not significantly altered in any experimental groups. Furthermore, total plasma protein synthesis increased in rats subjected to either starvation or hemorrhagic shock, but decreased in starved rats that were subsequently shocked.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effects of soluble and immobilized fibronectins on aortic endothelial cell proliferation and attachment.

We studied the effects of soluble and immobilized forms of plasma fibronectin on bovine aortic endothelial cell (AEC) proliferation and attachment. Soluble fibronectin stimulated AEC growth at 10 micrograms/ml, but at higher concentrations of soluble fibronectin AEC growth was progressively inhibited. The growth rates of arterial smooth muscle cells (ASMC) and dermal fibroblasts (DF) were not altered by soluble fibronectin concentrations of 10 to 100 micrograms/ml. Plasma fibronectin, immobilized by attachment to culture dish surfaces, had no significant effects on the proliferation of any of the cell types examined. The attachment rates of AEC were decreased in the presence of 50 micrograms/ml soluble fibronectin. Immobilized fibronectin increased the rate of AEC attachment, but had no significant effects on ASMC or DF attachment; however, 12 h after plating there was nearly 100% attachment in all groups, whether or not fibronectin was present in the system. That soluble and immobilized fibronectins elicit disparate cellular responses is consistent with published reports of different cell surface receptors for different forms of the protein; in this manner, cells enmeshed in an interstitial matrix containing immobilized fibronectin could still respond to soluble fibronectin in the extracellular milieu.

Quantitative evaluation of surgical task performance by remote-access endoscopic telemanipulation.

BACKGROUND: The performance limitations inherent in minimally invasive surgery may be overcome by using an interface that provides intuitive orientation for video display and tool manipulation. A prototype remote-access endoscopic telemanipulator was designed to fulfill these requirements and used for a surgical anastomosis task. METHODS: A remote-access telemanipulator system, employing remote center-of-motion geometry, was used to complete distant in vitro tubular anastomoses. The performance of four surgeons using this system was compared with that achieved in completing the same anastomosis task in an open environment using open surgical techniques and in a minimally invasive environment using standard laparoscopic methods. RESULTS: The average performance times for completion of the anastomosis task was 1448 +/- 130 s using the telemanipulator system compared with 2108 +/- 291 s with laparoscopic instruments and 296 +/- 25 s with conventional techniques. Leakage rates from the tubular anastomoses were 5.2 +/- 1.4 ml/s in the telemanipulator group, 6.9 +/- 2.0 ml/s in the laparoscopic group, and 3.2 +/- 0.9 ml/s in the conventional methods group. All experimental subjects were able to complete the assigned task in each experimental condition successfully without complications. CONCLUSIONS: Our results in this pilot study suggest that remote-access endoscopic telemanipulation can execute complex three-dimensional manipulations, and that the intuitive orientation of the surgeon's workstation may contribute to easier task completion.

Altered characteristics of B16 melanoma cells induced by chemically crosslinking fibronectin to cell surfaces.

The loss of fibronectin from tumor cell surfaces has been correlated with an increased incidence of metastases. To determine directly whether cell surface fibronectin influences the metastatic potential of solid tumors, we chemically crosslinked fibronectin to B16 murine melanoma cells using a photosensitive heterobifunctional crosslinking reagent, N-succinimidyl-4-azidophenyl-1,3 dithiopropionate (SADP). Cell attachment to plastic surfaces was increased in cells to which fibronectin was attached; cell growth over a 24-hr period was not significantly affected by the addition of fibronectin. When C57BL/6 mice were injected with fibronectin-crosslinked B16 cells, there was a 63% reduction in the number of pulmonary nodules compared to untreated controls. These results are consistent with the hypothesis that fibronectin enhances the recognition and removal of tumor cells from the circulation, possibly by cells of the reticuloendothelial system.

Assessment of popliteal arterial occlusive disease with 2D time-of-flight MRA.

OBJECTIVE: The purpose of this study was to evaluate both morphology and blood flow in peripheral arteries with occlusive lesions using MR angiography (MRA) and velocity-encoded cine MRI. MATERIALS AND METHODS: Two-dimensional time-of-flight MRA and velocity-encoded cine MRI were performed in nine patients with peripheral arterial occlusive disease. Findings on MR angiograms were verified by conventional angiography. RESULTS: All the stenotic lesions in the popliteal arteries were depicted by MRA. The degree of the stenoses in the artery was overestimated by MRA. Major collateral circulations were demonstrated. Velocity-encoded cine MRI provided flow velocity information on the arteries above and below the stenoses. The flow velocity waveform was monophasic above and below the stenosis. The peak systolic velocity in the artery below the stenosis was reduced compared with that above the stenosis (p < 0.05). CONCLUSION: The combination of MRA and velocity-encoded cine MRI has clinical potential for the evaluation of peripheral arterial occlusive disease.