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S K Williams

Publications and source records attributed to S K Williams.

At least 91 records · Page 5Linked to original sources
PubMed1988

Kinetics of endothelial cell-surface attachment forces.

Physical and biochemical forces exist that are necessary for the persistent attachment and function of ECs on native and prosthetic blood vessels. The optimization of conditions that permit regeneration of these attachment forces may allow rapid establishment of a durable, biocompatible EC monolayer. We examined the effects of three major factors, protein substrate, EC incubation time, and shear stress, on the attachment kinetics of human adult ECs to two different polymers. ECs were incubated up to 30 minutes on polymers (PS or PET) coated with extracellular matrix proteins: collagen I/III, fibronectin, collagen IV/V, laminin, gelatin, or saline control. After incubation, continued attachment in the presence of shear stress (created in a rotating disc device) between zero and 90 dynes/cm2 for 30 minutes was evaluated. Maximal adherence was observed on all substrates by 30 minutes. Therefore, after a 30-minute incubation, the percentage of cells attached (postshear ECs/preshear ECs/preshear ECs X 100) was measured as a function of shear stress. ECs attached to a matrix of fibronectin or collagen I/III demonstrated shear-resistant adherence after as little as 5 minutes of static incubation before initial shear exposure. By 30 minutes, more than 90% of the ECs on both matrices demonstrated the ability to remain attached in the presence of 90 dynes/cm2 of shear stress. We conclude that forces that attach ECs to surfaces are affected by temporal factors (incubation time) and substrate composition and may be quantified with a defined shear stress detachment assay. Understanding and manipulating these temporal physiochemical parameters should allow one to re-create an optimal EC monolayer on a blood-contacting surface.

Adhesiveness↗
PubMed1988

Quantitative analysis of random motility of human microvessel endothelial cells using a linear under-agarose assay.

Angiogenesis is a multistep process intimately involved in embryonic development and subsequent cardiovascular homeostasis and pathology. A major event in the process of angiogenesis is endothelial cell migration. Common in vitro assays (filter, under-agarose, phagokinetic track) used for the evaluation of migration are interpreted by measurements such as leading front distance, total cells migrated, and total area of migration. However, these quantities depend very heavily upon the physical aspects of the assay such as geometry, chemoattractant concentration and diffusivity, and observation time. Thus, while these common cell motility measurements are convenient, they do not represent solely the intrinsic cell response to an attractant. Alternatively, cell motility responses can be described by parameters which do not depend on the physical aspects of the assay system. Such parameters, termed phenomenologic parameters, have been defined for cell migration in a mathematical model derived by others. This model defines two parameters, the random motility coefficient, mu, and the chemotaxis coefficient, chi, which describe the migration responses to uniform concentrations and to gradients of stimulant, respectively. We have used this approach to evaluate the random motility response of human microvessel endothelial cells isolated from omental fat. Human microvessel endothelial cell random motility was measured in uniform concentrations of heparin (10(-3) to 10(3) micrograms/ml) using an under-agarose assay with linear geometry. The value of mu was found to remain constant at 8.2 x 10(-9) cm2/second for all concentrations tested and without heparin. These data indicate that heparin at these concentrations does not significantly stimulate random migration of human microvessel endothelial cell. These results suggest that the potentiating effect of heparin on angiogenesis may not be mediated through a direct affect on endothelial cell migration. Because the random motility coefficient and chemotaxis coefficient are representative of intrinsic cell motility behavior, their use should provide more specific information on endothelial cell migration than other commonly used measurements.

Cell Division↗
PubMed1987

Mechanism of sucrose uptake by isolated rat hepatocytes.

The transport of molecules by nonspecific endocytosis has been described in many cell types, but it has not been characterized in hepatocytes. Because of its central role in the clearance of solutes from portal blood, endocytosis might represent a significant mode of cellular transport. We investigated the mechanism of sucrose uptake in an isolated hepatocyte system. Liver cells were isolated by perfusion and collagenization of rat liver, followed by differential centrifugation. Hepatocytes were then incubated with 14C-sucrose and harvested by spinning through oil in microfuge tubes. Radioactivity was standardized against DNA content. We found that sucrose uptake is concentration-dependent from 5 microM to 100 mM and follows first-order kinetics. Washout studies indicate that exocytosis is responsible for the dynamic equilibrium reached. Arrhenius analysis of temperature dependence yields a linear plot (Ea = 14.2 Kcal/mol). In addition, sucrose uptake is independent of cellular ATP levels. We conclude that sucrose is transported by fluid-phase micropinocytosis in isolated hepatocytes and that this transport mechanism may be important in the uptake of diverse molecules into liver cells.

Adenosine Triphosphate↗
PubMed1987

Initial adherence of human capillary endothelial cells to Dacron.

Successful endothelialization of vascular grafts by seeding with endothelial cells (EC) at implantation is related to the number of EC which initially adhere to the graft. Using an in vitro system we examined the initial adherence of EC from human perinephric fat capillaries to woven Dacron that was either unmodified or precoated with several substrates. We studied capillary EC because they have not been investigated as a source of EC for graft seeding, although transinterstitial capillary ingrowth is one possible mechanism for spontaneous graft endothelialization. EC were isolated using collagenase and characterized morphologically and functionally including positive factor VIII-related antigen staining. EC were studied at three phases in culture: (A) primary EC with no subcultivations (EC-0); (B) EC after two subcultivations with trypsin (EC-2); and (C) EC after 10 subcultivations with trypsin (EC-10). EC were seeded onto graft material at a density of 10(5) cells/cm2 (100% confluence) and examined for cell counts and morphology after one day in culture by light and electron microscopy. Results are as follows: (table; see text) The conclusions are: (1) All capillary EC demonstrated adherence to Dacron, but this initial adherence was strongly influenced by graft pretreatment with collagen or plasma. (2) Serially subcultivated EC (EC-2 and EC-10) had significantly higher initial adherence to pretreated Dacron compared to the primary cells (EC-0) (P less than .05). This suggests that briefly cultured and subcultivated EC have superior initial adherence characteristics to treated dacron compared to primary EC with no subcultivations. (3) Fat capillary EC are easily procured and cultured and provide a rich source of human EC for endothelializing vascular prostheses.

Blood Vessel Prosthesis↗
PubMed1987

Cutaneous application of safflower oil in preventing essential fatty acid deficiency in patients on home parenteral nutrition.

Essential fatty acid deficiency (EFAD) is observed in patients with massive bowel resection who are placed on home parenteral nutrition (HPN). We investigated the use of cutaneously applied safflower oil to prevent EFAD. Five subjects on HPN supplemented with intravenous (IV) fat emulsions underwent a three-phase study: 1) no IV fat emulsions for 4 wk; 2) cutaneous safflower oil for 4-6 wk; 3) oral safflower oil for 4 wk. Fatty acid profiles (FAP) of plasma were obtained during each phase. Significant decreases in linoleic and arachidonic acid occurred by the end of phase 1 and the triene:tetraene ratio rose from a baseline value of 0.1 to 0.5. This ratio returned to 0.2 by the end of phase 2 and significant increases in linoleic and arachidonic acid occurred. Only one of five subjects completed the oral phase (3). Cutaneous safflower oil may improve plasma FAP but adequacy of tissue stores remains unanswered. Liver function tests need to be monitored if this treatment modality is utilized.

Administration, Cutaneous↗
PubMed1986

Use of an endothelial monolayer on a vascular graft prior to implantation. Temporal dynamics and compatibility with the operating room.

The temporal sequence of events was examined from initial contact of endothelial cells (ECs) to Dacron until the establishment of a monolayer. Cultured human adult ECs were radiolabeled, seeded onto Dacron, and adherence was quantified after vigorous washing. Firm adherence of 70% of the seeded ECs was seen by 2 hours to untreated Dacron, by 30 minutes to Dacron pretreated with a combination of interstitial type I/III collagen and an amnion-derived basement membrane (Type IV) collagen surface, and by 10 minutes to plasma-coated Dacron. Parallel samples were examined morphologically by scanning electron microscopy (SEM) to evaluate the adherence of ECs to surfaces. ECs seeded onto plain Dacron exhibited limited adherence, while cells on plasma-treated Dacron exhibited limited cell-cell associations. On basement membrane-treated Dacron, by 30 minutes the ECs exhibited a flat attenuated morphology, completely covering the graft surface. This time-frame is compatible with most vascular procedures, making an immediately endothelialized graft feasible.

Arterial Occlusive Diseases↗
PubMed1986

Use of freshly isolated capillary endothelial cells for the immediate establishment of a monolayer on a vascular graft at surgery.

Endothelial seeding of vascular graft surfaces may lead to a less thrombogenic surface. We examined the feasibility of using microvessel endothelial cells derived from human fat for seeding purposes. Human fat was treated with collagenase for 24 minutes, washed, and purified in a Percoll gradient separation. This yielded 1.25 +/- 0.45 X 10(6) cells/gm of fat. After a 1-hour incubation on plasma-coated Dacron, 2.8 +/- 1.5 X 10(4) cells remained firmly adherent to the surface. When exposed to flow for 2 hours at a shear stress of 0 to 80 dyne/cm2, between 50% and 100% of the initially adherent cells remained adherent. Statistical analysis of this data failed to demonstrate a strong relationship between the number of adherent cells and the shear rate. Scanning electron microscopy demonstrated endothelial cells in various stages of attachment to the plasma-coated Dacron. Although most cells were still round and only focally attached to the surface, some cells were maximally flattened, forming cell-to-cell contact. Because of the high cell yield and the firm adherence characteristics, we conclude that microvessel endothelial cells may offer the possibility for confluent endothelial cell seeding of a graft at the time of surgical implantation without the need for cell culture.

Adipose Tissue↗
PubMed1985

Morphometric evaluation of diabetes-associated ovarian atrophy in the C57BL/KsJ mouse: relationship to age and ovarian function.

Progressive, diabetes-associated ovarian atrophy was analyzed in C57BL/KsJ diabetic (db/db) and control (+/?) mice between 2 and 16 weeks of age. Tissue changes were histologically and morphometrically analyzed and compared with ovarian functional indices (i.e., serum estradiol and progesterone) and metabolic (i.e., glucose uptake and estradiol sequestration) parameters. No significant differences were found between the ovarian follicular populations of either group at 2 and 4 weeks of age. However, between 4 and 8 weeks, the ovaries of diabetic mice exhibited marked stromal and follicular degeneration and an associated decline in the population of viable follicles as compared with controls. Between 8 and 16 weeks of age the follicular atrophy in the diabetics became more marked, as compared with controls, with the accumulation of intracellular lipid pools accenting the tissue degeneration and adiposity observed in both follicular and stromal compartments. In addition, ovarian function was depressed after 6 weeks of age in diabetic females as compared with controls as indicated by lowered serum estradiol and progesterone levels. Ovarian glucose uptake was enhanced in diabetic females while the ability of the ovary to sequester radiolabeled estradiol declined between 4 and 16 weeks of age as compared with controls. These data indicate that ovarian dysfunction in the (db/db) mutant mouse is associated with follicular atrophy, adiposity, impaired steroidogenesis, and imbalanced glucose utilization. These events occur in temporal association with the onset and progressive exacerbation of the hyperglycemic condition. It is suggested that ovarian involution in these mutants is directly related to an impaired follicular ability to metabolize properly the elevated intracellular glucose concentrations that develop in the (db/db) mice as compared with controls.

Aging↗
PubMed1985

Insulin binding and vesicular ingestion in capillary endothelium.

Capillary endothelium can actively regulate vascular permeability of various serum proteins. Hormones such as insulin must interact with this capillary barrier in order to reach their respective target tissues. We have studied the binding and subsequent internalization of 125I-insulin in both native (freshly isolated) and primary cultured capillary endothelium derived from rat epididymal fat pads. Insulin association with the endothelium, internalization and degradation differed between freshly isolated and primary cultured capillaries. Specific binding in freshly isolated and cultured capillaries was temperature dependent, and was competitively inhibited in the presence of unlabelled insulin. Primary cultures of capillaries grown to confluence did not exhibit specific binding of insulin. Despite the lack of specific receptors for insulin, cultured cells vesicularly internalized insulin. Greater than 50% of the total associated insulin was not degraded by cultured endothelium. Morphological examinations using ferritin labelled insulin localized insulin associated to the capillary endothelial cell membrane and sequestered within pinocytotic vesicles. Incubation of freshly isolated capillaries with insulin stimulated the fluid phase endocytosis of 14C-sucrose; however, insulin had no effect on fluid phase endocytosis in cultured capillaries. These results indicate that capillary endothelium, isolated from rat epididymal fat, exhibit specific receptors for insulin. Binding of insulin to the capillary membrane is followed by internalization into cytoplasmic vesicles and partial degradation.

Animals↗
PubMed1985

Endothelialization of human collagen surfaces with human adult endothelial cells.

Artificial vascular grafts are currently used to restore blood flow to ischemic tissue. Although the long-term patency of large diameter grafts is relatively acceptable, small diameter (less than 4 mm) grafts exhibit poor long-term patency rates. One technique to create a nonthrombogenic surface on artificial prostheses has been to seed with endothelial cells derived from autologous vessels. We have examined the interaction of human adult endothelial cells with the natural collagen surfaces presented by human amnion. Scanning electron microscopic evaluation revealed that human adult endothelial cells adhered rapidly to both the basement surface (collagen types IV and V) and interstitial surface (collagen types I and III) of amnion. However, the adherence of cells was significantly greater on the basement membrane surface. In addition, human adult endothelial cells rapidly formed close cell-to-cell interactions on basement membrane as compared with cells seeded onto the interstitial surface. These results suggest that seeding of endothelial cells onto artificial surfaces will be facilitated if the surface simulates the natural basement membrane to which endothelial cells natively adhere.

Adult↗
PubMed1985

Adult human endothelial cell compatibility with prosthetic graft material.

We have developed a system for the in vitro evaluation of the interaction of human adult endothelial cells (HAEC) with prosthetic vascular graft material. HAEC, isolated from adult human iliac veins, proliferated vigorously in culture for approximately 70 population doublings. The large number of HAECs produced permitted high-density seeding of prosthetic grafts. Samples of prosthetic material were immobilized on a plastic ring and were used either untreated or coated with extracellular matrix, fibronectin, or plasma. HAEC were seeded at high density and adherence was evaluated by light and electron microscopy after a 2-hr incubation. While essentially no HAEC adhered to untreated grafts, treatment of grafts with either extracellular matrix, plasma, or fibronectin resulted in dramatic adherence of HAEC. The highest density of HAEC adherence was observed on collagen-coated Dacron grafts, and was equal to the cell density observed in confluent monolayers of HAEC grown on gelatin-coated tissue culture plastic. This study demonstrates a method capable of determining HAEC-graft biocompatibility prior to the use of an in vivo system.

Adult↗
PubMed1985

Preferential transport of non-enzymatically glucosylated ferritin across the kidney glomerulus.

We have examined the significance of carbohydrate structure to the transglomerular passage of proteins. Carbohydrate-free (non-glycosylated) ferritin, prepared by Concanavalin-A-sepharose affinity chromatography, was perfused into rat kidneys, and was observed to be restricted from transglomerular transport and to accumulate within the lamina rara interna of the glomerular basement membrane. Visibility of the laminar structure of the glomerular basement membrane was enhanced following perfusion fixation containing tannic acid, permitting the observation of charge dense regions within the basement membrane. Non-enzymatically glucosylated ferritin was not restricted by the lamina rara interna and was observed to penetrate the lamina densa and lamina rara externa. Glucosylated ferritin was observed to be sequestered also by epithelial pinocytic vesicles and to be accumulated within multivesicular bodies. Quantitative measurements using fluorescently labelled ferritins indicated the preferential clearance of glucosylated ferritin from the plasma and preferential appearance of glucosylated ferritin in the urine. The differential transport of glucosylated ferritin was not due to the formation of a cationic protein, as isoelectric focusing established that glucosylation of ferritin results in a more anionic protein. These studies suggest that glucosylation of anionic proteins results in their increased transglomerular permeability. This increased protein permeability could contribute to the proteinuria observed in diabetic microangiopathy.

Animals↗
PubMed1984

Endocytosis and exocytosis of protein in capillary endothelium.

The transport of proteins across continuous capillary endothelium is believed to be mediated by micropinocytic vesicles which shuttle molecules between the lumenal and abluminal plasma membrane. We have studied the ability of capillary endothelial cells isolated from rat epididymal fat to endocytose fluorescently labelled ovalbumin within micropinocytic vesicles. Net association of fluorescent ovalbumin with endothelial cells reaches an equilibrium after 40 minutes of incubation. This equilibrium is presumably due to a balance between endocytosis and subsequent exocytosis of this protein. Capillaries equilibrated with fluorescent ovalbumin exhibited rapid exocytosis of this protein when it was removed from the external medium. The rate of endocytosis was concentration dependent and obeyed the kinetics expected for adsorptive phase endocytosis. High concentrations of ovalbumin stimulated the ingestion of 14C-sucrose, a marker of fluid endocytosis, suggesting that protein can affect the movement of vesicles within the endothelial cytoplasm. These results imply that capillary endothelium isolated from rat epididymal fat exhibits the ability to endocytose and subsequently exocytose protein. This demonstrates that the two components of endothelial vesicular transport or transcytosis can be observed and studied in a system of isolated capillary endothelium.

Animals↗
PubMed1984

Enhanced vesicular ingestion of nonenzymatically glucosylated proteins by capillary endothelium.

Isolated capillary endothelial cells exhibit an enhanced vesicular ingestion of nonenzymatically glucosylated myoglobin and ovalbumin. Proteins were incubated in the presence of glucose and the extent of nonenzymatic glucosylation was assessed using NaB3H4 reduction. Unmodified and glucosylated proteins were fluorescently labeled, and vesicular ingestion was quantified using a system of isolated capillary endothelium. Glucosylated myoglobin exhibited a 3.5-fold greater rate of vesicular injection as compared to unmodified myoglobin during the initial 30 min of ingestion. Glucosylated ovalbumin was vesicularly ingested at a rate 15-fold greater than the rate observed for unmodified ovalbumin during the initial 30 min of ingestion. These results indicate that glucosylation of small proteins may alter their subsequent recognition by the endothelial cell plasma membrane and result in increased sequestration within micropinocytic vesicles.

Animals↗
PubMed1984

The preparation and use of fluorescent-protein conjugates for microvascular research.

A procedure is described for making large quantities (100 ml) of fluorochrome-labeled albumin. Chromatographic techniques are described for the purification of commercial albumin (BSA) and the purification of albumin from serum. We report experimentally determined optimal conditions for the covalent attachment of fluorescent dyes (rhodamine isothiocyanate (RITC) and fluorescein isothiocyanate (FITC] to albumin. Subsequent removal of all unreacted fluorescent material (UFM) was achieved using charcoal adsorption. We observed no loss of protein following charcoal treatment. The final protein conjugate was analyzed by polyacrylamide gel electrophoresis, gel chromatography, and isoelectric focusing. The conjugates were determined to be free of UFM and homogeneous with respect to molecular weight. However, FITC conjugation lowered the average isoelectric point of albumin by 0.1 to 0.3 pH units. Illustrations of combining fluorescence microscopy with FITC-BSA and RITC-BSA to view microvascular phenomena in skeletal muscle and the heart are given. Knowledge of the biochemical characteristics of the fluorochrome employed is important for proper interpretation of experimental results using this technique.

Animals↗
PubMed1984

Glucose utilization by the mouse brain: influence of age and diabetes.

The effects of diabetes on the age-related changes in glucose utilization by various brain regions were examined in genetically diabetic (db/db) and normal (+/?) C57BL/KsJ mice following a 10 muCi injection of [3H]2-deoxyglucose (2-DOG). Brains were collected from 2 to 16-week-old mice at 30 min postinjection with 2-DOG, and the brain regions isolated by microdissection. Glucose utilization was expressed as mumol/mg/30 min for each brain region from match-paired control +/? and db/db mice. No differences in utilization were seen in brain regions from control and db/db mice at 2 weeks of age. In contrast, the diabetic condition effectively depressed the age-related increase in glucose metabolism associated with maturation in control mice between 4 and 16 weeks of age. Of particular interest was the observation that the pituitary gland of the db/db mice did not demonstrate a maturation-associated increase in glucose utilization typical of +/? mice. By 16 weeks of age, all of the brain regions of db/db mice exhibited a depressed glucose utilization rate as compared with +/? mice. These studies demonstrate that the diabetic condition impairs the normal age-related increase in CNS glucose utilization in the mouse, and suggests that decreased glucose utilization may be causally related to diabetes-associated, CNS and peripheral neuropathy.

Age Factors↗