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Biomedical subjects

S K Williams

Publications and source records attributed to S K Williams.

At least 55 records · Page 3Linked to original sources
PubMed1995

Interaction of colloidal gold-labelled glucosylated albumin with endothelial cell monolayers: comparison between cryofixation and glutaraldehyde fixation.

Bovine aortic endothelial cells (BAEC) were exposed to glucosylated albumin-gold complexes (GgA), and the distribution of the tracers was compared after cryofixation and after glutaraldehyde fixation. Morphometric analysis revealed differences in the GgA distribution depending upon the method of fixation used. In BAEC monolayers cryofixed after 3 min of incubation with GgA, tracer was observed in predominately apically located vesicular elements. After 16 min of incubation, all vesicular elements were labelled, and multivesicular bodies were the prominent labelled structure. In contrast, chemically fixed monolayers exhibited a heterogeneous distribution of GgA within vesicular profiles after 3 min and 16 min of GgA incubation. The differences in tracer distribution depending upon the fixation method must be resolved before the mechanism of vesicle-mediated endothelial cell transport function is defined and universally accepted.

Animals↗
PubMed1995

Isolation and culture of human trabecular meshwork cells by extracellular matrix digestion.

Like corneal endothelial cells, human trabecular meshwork cells are believed to be of neural crest origin, but demonstrate physiological properties and an antithrombogenic surface similar to vascular endothelial cells. One current method for isolating trabecular meshwork cells utilizes the motile nature of these cells to migrate away from a trabecular meshwork explant in culture to more distal regions of the culture dish. This 'outgrowth' technique is limited in practice by the relatively small number of cells that migrate per explant per unit time, thus hindering the ability to gather sufficient numbers of cells for comprehensive experimentation. For this reason, we have modified an extracellular matrix digestion technique in current use for the isolation of microvascular endothelial cells to isolate human trabecular meshwork cells. This procedure is both efficient and rapid for isolating large numbers of trabecular meshwork cells and results in the availability of trabecular meshwork cells in sufficient quantities for subsequent experimentation.

Adolescent↗
PubMed1995

Early in reperfusion following myocardial ischemia, leukocyte activation is necessary for venular adhesion but not capillary retention.

OBJECTIVE: The pathobiology of leukocyte sequestration in the coronary microcirculation following ischemia is unclear. We examined the location(s) and persistence of leukocyte sequestration of unactivated and preactivated blood in the coronary microcirculation early during reperfusion following ischemia. METHODS: Isolated rat hearts were subjected to 30 min of 37 degrees C, no-flow ischemia. Hearts were initially reperfused with diluted whole blood containing fluorescent leukocytes (DWB*). At 5, 20, and 35 min of reperfusion (R), the deposition of leukocytes in the coronary capillaries and venules was observed directly using intravital fluorescence microscopy. Four groups were studied: a nonischemic control group (Gr I), and postischemic groups reperfused with DWB* treated with vehicle (Gr II) or preactivated with 10(-8) M N-formylmethionyl-leucyl-phenylalanine (fMLP) (Gr III) or 10(-6) M fMLP (Gr IV). RESULTS: At R5, postischemic reperfusion with unactivated blood caused a significant trapping of leukocytes in coronary capillaries (Gr I = 2.2 +/- 0.4 versus Gr II = 5.6 +/- 0.6 leukocytes per capillary field, P < 0.05). Hearts in Gr IV exhibited significantly greater leukocyte retention in capillaries compared to all other groups at R5 (R5, Gr IV = 8.8 leukocytes per capillary field, P < 0.05) and at R35. At R5, although more leukocytes were observed adhered to the venules in Gr II compared to Gr I, the difference was not statistically significant (Gr I = 1.7 +/- 0.7 versus Gr II = 3.4 +/- 0.5 leukocytes per 100 microns venule, P = 0.23). DWB* preactivated with the lower concentration of fMLP (10(-8) M) resulted in a significant increase in venular leukocyte adhesion at R5 compared to Gr I and Gr II (Gr III 6.1 +/- 0.5, P < 0.05). After 35 min of reperfusion, a greater percentage of leukocytes remained in the capillaries than in the venules. CONCLUSIONS: These direct observations suggest that early in reperfusion after ischemia, both leukocyte and endothelial activation are necessary for venular adhesion, but that ischemia-induced coronary microvascular alterations are sufficient to promote leukocyte retention in coronary capillaries. These results also indicate that during 35 min of reperfusion, the degree of leukocyte washout is greater in the venules than in the capillaries. These results suggest that the mechanisms contributing to leukocyte retention early in reperfusion following myocardial ischemia are, indeed, different in the capillaries and venules and that the mechanisms affecting retention in capillaries are more persistent than those in the venules.

Animals↗
PubMed1994

Microvascular endothelial cell sodding of ePTFE vascular grafts: improved patency and stability of the cellular lining.

Small diameter (< 6 mm) synthetic vascular grafts fail at a clinically unacceptable rate due in large part to their inherent thrombogenicity. The development of a new cellular lining on synthetic vascular grafts would most likely improve the patency rates observed for these grafts in small diameter positions. We have evaluated the use of endothelial cell transplantation to accelerate the formation of a cell lining using microvascular endothelial cells derived from canine falciform ligament fat. This source of fat is histologically similar to human liposuction fat and was isolated using a collagenase digestion technique identical to methods used for human liposuction fat microvessel endothelial cell isolation. The isolated fat endothelial cells were sodded onto 4 mm ePTFE grafts using pressure to force the cells onto the luminal surface. This pressure sodding method permitted cell deposition in less then 3 min. Sodded and control (non-cell-treated) grafts were implanted as interpositional paired grafts using end-to-end anastomoses in the carotid arteries of mixed breed dogs. Each dog therefore received a sodded graft on one side and a control graft on the contralateral side. After 12 weeks of implantation all control grafts were occluded while 86% of the cell-sodded grafts remained patent. Statistical evaluation of the data revealed a significant improvement in patency of cell sodded grafts (McNemar's chi 2 P = .02). Morphological evaluation of grafts explanted at 5, 12, 26, and 52 weeks following implantation revealed the presence of a cell lining on sodded grafts which remained stable for a period of at least one year. This new cell lining exhibited morphologic characteristics of a nonthrombogenic endothelial cell lining. The development of this new intima, evaluated 5 weeks-1 year after implantation, was not associated with a progressive intimal hyperplasia. From these data we conclude that microvessel endothelial cells derived from canine falciform ligament fat can be rapidly isolated using an operating room compatible method. Cell deposition on synthetic grafts is subsequently accelerated using a pressure sodding technique. A cellular lining forms on the inner surface and is associated with a statistically significant improvement in the function of sodded grafts in a canine carotid artery model.

Adipose Tissue↗
PubMed1994

Liposuction-derived human fat used for vascular graft sodding contains endothelial cells and not mesothelial cells as the major cell type.

PURPOSE: Endothelial cell transplantation has been suggested as a method to improve the patency of prosthetic grafts used for vascular reconstruction. A major technical concern of all cell transplantation studies has been the purity of cells in the primary isolate used for subsequent transplantation. Accordingly we have evaluated the cellular constituents of liposuction-derived human fat with immunocytochemistry and scanning electron microscopy. METHODS: Samples of liposuction-derived human fat were processed for immunohistochemistry and subsequently stained for the presence of von Willebrand factor (vWF), alpha-smooth muscle cell actin, cytokeratin (peptide 18), and the endothelial cell-specific marker EN4. We also performed histochemistry studies on the cells derived from this fat after collagenase dispersion of the liposuction far. RESULTS: Immunohistochemistry revealed that 86.1% of the cells in intact, liposuction-derived fat express vWF, whereas 5.7% of the cells exhibited alpha-smooth muscle cell actin, and 1.0% expressed the mesothelial cell-related antigen, cytokeratin peptide 18. Expression of EN4 was found in 89.6% of the cells counted in intact far. After digestion of fat with collagenase and centrifugal separation of adipocytes from vascular and stromal cells, the expression of vWF, alpha-smooth muscle cell actin, and cytokeratin was 77.5%, 5.8%, and 2.1%, respectively. EN4 expression was observed in 74.6% of the isolated cells. Thus most cells present in liposuction-derived fat, even before tissue digestion and cell isolation, were characterized as endothelium. Although other cells common to mesodermally derived tissue were identified (e.g., adipocytes, smooth muscle cells, and mesothelium), they represented a minor fraction of the total cells present. On isolation, the number of cells expressing vWF- and EN4-specific antigens was less than that observed in intact fat. CONCLUSIONS: This finding suggests that a portion of cells reacting with antibodies in situ lose vWF and EN4 staining during the isolation procedure. Unlike omentum, liposuction-derived fat predominantly contains adipocytes and endothelial cells. On digestion of liposuction-derived fat and separation of cells, vascular endothelial cells represent the major cellular component.

Adipocytes↗
PubMed1994

Origin of endothelial cells that line expanded polytetrafluorethylene vascular grafts sodded with cells from microvascularized fat.

PURPOSE: Cell transplantation onto prosthetic vascular grafts remains an attractive technique to reduce the thrombogenicity of polymeric materials. In this study we evaluated whether autologous cells isolated from falciform ligament fat and transplanted onto the lumenal surface of 4 mm expanded polytetrafluorethylene grafts were the same cells present on the surface of these grafts when they were explanted from canine carotid arteries 3 weeks after their implantation. METHODS: The fluorescent dye PKH-26 was used to label transplanted cells to evaluate their fate after implantation of grafts as carotid artery replacements. This fluorescent dye homogeneously labeled all cells in the primary cell isolate. RESULTS: In vitro studies indicated that dye labeling was nontoxic, as evidenced by the normal growth characteristics of fluorescently labeled cells compared with nonlabeled cells. Immunocytochemical analysis of microvascularized fat before cell isolation determined that approximately 90% of the cells stained positive for von Willebrand factor2. At the time of explant, seeded grafts exhibited a nonthrombogenic lumenal cell lining as evidenced by the lack of adherent platelets or fibrin. Cells on the lumenal surface of grafts exhibited PKH-26 fluorescence emission. In addition, these cells expressed von Willebrand factor and actively sequestered DiI-acetylated low-density lipoprotein. CONCLUSIONS: We conclude that sodding of prosthetic grafts with autologous microvascularized fat-derived cells results in the formation of an endothelial cell lining on the lumenal flow surface. These endothelial cells are the same cells placed on the lumenal surface of the graft at the time of initial cell transplantation. Finally, a confluent monolayer forms after high-density cell sodding by the process of cell adherence and spreading, without the need for cell proliferation.

Adipose Tissue↗
PubMed1994

Microvascular endothelial cell sodding of 1-mm expanded polytetrafluoroethylene vascular grafts.

The formation of an endothelial cell lining on the inner surface of polymeric grafts may reduce the inherent thrombogenicity of synthetic implants. Endothelial cell transplantation onto the luminal surface of grafts has been suggested as one method of creating new endothelial cell linings on grafts. The purpose of this study was to morphologically evaluate the very early events of healing (between 4 and 14 days) of 1-mm-internal-diameter expanded polytetrafluoroethylene (ePTFE) grafts that were treated with autologous microvessel endothelial cells at the time of graft implantation. We evaluated the development of new intimal linings in microvascular endothelial cell-sodded 1-mm ePTFE vascular grafts and compared their healing characteristics with non-cell-treated grafts by using a rat aortic graft model. Endothelial cells were isolated from intraperitoneal fat pads of female rats and transplanted onto the grafts by using a pressure sodding method. One-centimeter-long grafts were immediately implanted as interpositional grafts in the aorta. Non-cell-treated grafts were also implanted. Grafts were explanted 4, 7, and 14 days after implantation and were evaluated by light and scanning electron microscopy. Morphometric analysis of the graft surfaces revealed the cellular coverage in sodded grafts to be 93.7 +/- 8.7% and in nonsodded grafts, 1.1 +/- 1.9%. Areas not covered by cells exhibited thrombus and bare graft. The luminal lining of cells exhibited morphological characteristics, indicating they were antithrombogenic, based on morphological criteria, and exhibited characteristics of endothelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗
PubMed1993

Interferon-gamma-inducible endothelial cell class II major histocompatibility complex expression correlates with strain- and site-specific susceptibility to experimental allergic encephalomyelitis.

The interaction between encephalitogenic lymphocytes and the cerebral microvascular lining is considered to be an important initial step in the recruitment of immune cells into the central nervous system (CNS) under pathological conditions such as multiple sclerosis (MS) and its investigative analog, experimental allergic encephalomyelitis (EAE). This study was conducted in order to examine whether differences in microvascular endothelial cell expression of several molecules involved in lymphovascular interactions correlate with the strain and organ-specific development of EAE. Cerebral and epididymal microvascular endothelial cells (EC) were isolated from SJL and B10.S mice, which, despite MHC-compatibility (H-2S), differ in their ability to develop EAE. The subcultured cells were then analyzed by flow cytometry for their ability to express class I MHC, class II MHC and ICAM-1 molecules in response to treatment with murine recombinant interferon-gamma (IFN-gamma). Over a range of doses and times, cerebral EC cultures derived from EAE-susceptible SJL mice expressed two-fold higher levels and higher cell surface densities of class II molecules than cerebral EC cultures derived from EAE-resistant B10.S mice, whereas class I and ICAM-1 molecules were comparably upregulated on both SJL and B10.S cerebral EC. In contrast, both SJL and B10.S epididymal EC cultures expressed lower but comparable levels of class II molecules in response to IFN-gamma. Class I and ICAM-1 molecules, however, were upregulated to at least the same degree as that observed on cerebral EC derived from both strains.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Formation of a multilayer cellular lining on a polyurethane vascular graft following endothelial cell sodding.

Small-diameter (less than 6 mm) clinically available vascular grafts often fail due in part to the inherent thrombogenicity of artificial polymers. Transplantation of endothelial cells onto the lumen of these vascular grafts has been suggested as one method to overcome this thrombogenicity. We have developed a compliant polyurethaneurea (PEUU) 4-mm graft with a luminal surface modified by a glow discharge gas plasma. Autologous microvessel endothelial cells were isolated from canine falciform ligament fat, were transplanted onto the luminal surface of the grafts using an intraoperative isolation and sodding technique, and both endothelial-cell-treated and non-cell-treated grafts were placed as bilateral carotid interposition grafts in a canine model. After 5 weeks of implantation, explanted control (non-cell-treated) grafts exhibited a deposition of platelets, white cells and fibrin characteristic of a thrombogenic surface. MVEC sodded grafts exhibited a multicellular lining within but distinct from the lumen of the PEUU graft. The blood-contacting surface of this lining exhibited an antithrombogenic endothelial cell monolayer. We suggest that the PEUU graft supported the initial deposition of MVEC and development of and endothelial cell lining. During the 5 weeks of implantation this lining continued to proliferate and detached from the PEUU graft substratum. The final neocellular lining exhibited a luminal diameter and histological features similar to a native artery.

Animals↗
PubMed1992

Effects of growth hormone-releasing factor and vasoactive intestinal peptide on proliferation and steroidogenesis of bovine granulosa cells.

Recent studies have suggested that growth hormone-releasing factor (GRF), like vasoactive intestinal peptide (VIP), may enhance follicle-stimulating hormone (FSH)-stimulated steroidogenesis in cultured rat granulosa cells (GC). Because effects of GRF or VIP on GC proliferation have not been reported, we evaluated and compared the effect of GRF to that of VIP using cultured bovine GC. Undifferentiated GC from 1-5 mm bovine follicles were established for 2 days in medium containing 10% fetal calf serum, washed and then cultured in chemically defined medium for an additional 2 days. Two-day treatment with 2.5-1000 ng/ml of VIP had no effect (P greater than 0.05) on proliferation or progesterone production of bovine GC in the presence or absence of 200 ng/ml FSH. In comparison, 100, 250, 500, 1000 or 2000 pg/ml of human [desNH2Tyr1,D-Ala2,Ala15]-GRF(1-29)-NH2 analog caused a dose-dependent stimulation (P less than 0.05) of GC proliferation in the absence and presence of 5 micrograms/ml insulin. However, the GRF analog had no effect (P greater than 0.05) on GC progesterone production (expressed as ng/10(5) cells/24 h) in the absence or presence of 5 micrograms/ml insulin. The effects of GRF analog on progesterone production and cell proliferation were not influenced by co-culture with 200 ng/ml FSH. GRF(1-44)-NH2 also stimulated cell proliferation but had no effect on basal or FSH-induced progesterone production. These results suggest that GRF may play a role in GC proliferation during follicular development in the bovine.

Animals↗
PubMed1992

A porcine model for adipose tissue-derived endothelial cell transplantation.

The transplantation of endothelial cells represents a technology which has been suggested for applications ranging from improvement in function of implanted vascular devices to genetic therapy. The use of microvascular endothelial cell transplantation has seen increased use both in animal studies as well as clinical use. This report describes our techniques for the isolation and establishment of initial cultures of microvascular endothelial cells derived from porcine fat. A variety of anatomic sites within the pig were evaluated to determine the appropriateness of different sources of fat for endothelial cell isolation. The properitoneal fat was determined to be optimal due to the predominance of endothelium in this tissue and the ease of isolation of microvascular endothelium following collagenase digestion. The study of endothelial cell transplantation in the porcine model is now possible using the methods described for adipose tissue-derived microvessel endothelial cell isolation.

Actins↗
PubMed1991

Formation of a functional endothelium on vascular grafts.

The lack of a functional endothelial cell lining on artificial polymeric vascular grafts severely reduces their effectiveness in replacing small caliber (less than 6 mm) blood vessels. Techniques have now been developed to transplant autologous endothelial cells from one site in the body onto the surface of grafts prior to implantation. Pre-clinical animal trials provide evidence that grafts sodded with autologous, fat-derived, microvessel endothelial cells exhibit a stable, antithrombogenic lining of endothelium. The new endothelial cell lining exhibits morphologies identical with endothelium on native blood vessels. The effectiveness of endothelial cell sodding techniques in pre-clinical animal trials provides support for expanded clinical trials.

Animals↗
PubMed1991

Cultured and immediately procured endothelial cells: current and future clinical applications.

Great progress has been made in the last several years in our ability to culture human endothelial cells. In addition, techniques to immediately procure and utilize these cells have also been developed. The purpose of this paper is to present an overview of the current and potential uses of these cells in both vascular and nonvascular conditions. It is likely that endothelial cells will be used in a variety of applications in the near future. Immediately procured and cultured cells will be used to resurface vascular prosthetic grafts. They may also be used on the surface of vessels following procedures such as balloon angioplasty or atherectomy. In addition, they may be placed upon the surface of implantable devices such as expandable stents. Through the mechanism of genetic engineering, these cells may be modified to produce proteins, which may modify thrombogenicity and perhaps decrease the rate of recurrent stenosis by influencing cellular hyperplasia. Genetically modified endothelial cells also have great potential in nonvascular disease. Their contact with circulating blood makes them an ideal cell for production of proteins to correct systemic conditions such as the insulin deficiency found in diabetes mellitus. The application of endothelial cell biology in both vascular and nonvascular science represents one of the most exciting fields of research active today.

Blood Vessel Prosthesis↗
PubMed1991

Origin of cells that line damaged native blood vessels following endothelial cell transplantation.

Endothelial cell (EC) transplantation has been proposed as a method to reduce the thrombogenicity of both vascular grafts as well as injured native blood vessels. While techniques have been developed to establish EC monolayers on these surfaces, a major question that remains is whether the cells that exist on the blood flow surface are the same cells placed on the surface at the time of transplantation. We have developed an intravital fluorescent staining technique that permits isolated, autologous, fat-derived microvascular endothelial cells (MVEC) to be labeled and subsequently detected following their transplantation. In our study, rat abdominal aortas (AA) were injured with a 3F embolectomy catheter, and the injured surfaces were immediately treated with fluorescently labeled MVEC. Five days after transplantation, AA were evaluated by both scanning electron and fluorescence microscopy. Results of scanning electron microscopy showed the existence of nonthrombogenic regions in the areas of injury, and fluorescence microscopy of the identical areas established that these cells contained fluorescent dye. Our results indicate that the cells that line these injured areas of native vessels are the same cells that were originally transplanted. Our intravital fluorescence technique provides a method to trace the origin and disposition of transplanted cells on the vascular surfaces.

Adipose Tissue↗
PubMed1991

Microvessel derived endothelial cell isolation, adherence, and monolayer formation for vascular grafts.

MVECs can be isolated from animal and human fat in quantities and in a pure enough form to produce a cell-lined vascular graft. In animal studies grafts treated with these cells are associated with prolongation of graft patency. In human implants definitive expression factor VIII related antigen cells has been performed on an MVEC treated graft at 9 months. Further studies will be necessary to provide further insight into the healing and long-term behavior of these grafts, both with respect to the thrombogenicity of the surface as well as the influence on anastomotic hyperplasia.

Animals↗
PubMed1991

Optimization of human endothelial cell attachment to vascular graft polymers.

Endothelial cells (EC) covering the blood-contacting surface of a prosthetic material could potentially enhance the subsequent nonthrombogenicity of the surface. In order to create such a surface, the EC must become attached to the surface, spread and ultimately form a monolayer. In this study we examined several factors that influence these processes. On ePTFE surfaces, surface pretreatment with human serum for 30 minutes at a concentration of 1.4 gm percent protein resulted in significantly more attached EC when compared to other concentrations or when compared to fetal calf serum or human serum albumin. The rate of EC spreading was strongly influenced by temperature, with a maximum occurring at 37 degrees C. During real-time video microscopy, it was noted that the rate of EC attachment and spreading was primarily dependent on arrival of the EC to the surface rather than attachment and spreading. Thus as a method of increasing EC delivery, the concept of filtering EC onto the graft lumenal surface was tested by pressurizing the graft lumen to speed EC delivery to the surface. This technique produced a 2 to 5-fold increase in EC attachment when compared to gravity forced cell deposition. We conclude that an ePTFE graft can be rapidly endothelialized using these simple measures.

Biocompatible Materials↗