The effect of combined arterial hemodynamics on saphenous venous endothelial nitric oxide production.

INTRODUCTION: Evidence exists that an ideal bypass conduit should have a functional endothelial cell surface combined with mechanical properties similar to those of native arteries. We hypothesized that the effect of combined arterial levels of pulsatile shear stress, flow, and cyclic strain would enhance saphenous venous endothelial cell nitric oxide (NO) production, and that variations in these "ideal" conditions could impair this function. We studied NO production as a measure of endothelial function in response to different hemodynamic conditions. METHODS: Human adult saphenous venous endothelial cells were cultured in 10-cm silicone tubes, similar in diameter (5 mm) and compliance (6%) to a medium-caliber peripheral artery (eg, popliteal). Tube cultures were exposed to arterial conditions: a combined pressure (120/80 mm/Hg; mean, 100 mm/Hg), flow (mean, 115 mL/min) and cyclic strain (2%), with a resultant pulsatile shear stress of 4.8 to 9.4 dyne/cm2 (mean, 7.1). Identical tube cultures were used to study variations in these conditions. Modifications of the system included a noncompliant system, a model with nonpulsatile flow, and a final group exposed to pulsatile pressure with no flow. NO levels were measured with a fluorometric nitrite assay of conditioned media collected at 0, 0.25, 0.5, 1, 2, and 4 hours. Experimental groups were compared with cells exposed to nonpulsatile, nonpressurized low flow (shear stress 0.1 dyne/cm2) and static cultures. RESULTS: All experimental groups had greater rates of NO production than cells under static conditions (P <.05). Cells exposed to ideal conditions produced the greatest levels of NO. Independent decreases in compliance, flow, and pulsatility resulted in significantly lower rates of NO production than those in the group with these conditions intact (vs noncompliant P <.05, vs nonflow P <.05, and vs nonpulsatile P <.05). CONCLUSIONS: Our results show that in the absence of physiologically normal pulsatility, cyclic strain, and volume flow, endothelial NO production does not reach the levels seen under ideal conditions. Pulsatile flow and compliance (producing flow with cyclic stretch) play a key role in NO production by vascular endothelium in a three-dimensional hemodynamically active model. This correlates biologically with clinical experience linking graft inflow and runoff and the mechanical properties of the conduit to long-term patency.

Shear stress decreases endothelial cell tissue factor activity by augmenting secretion of tissue factor pathway inhibitor.

Monolayers of human umbilical vein endothelial cells were activated with 50 U/mL interleukin-1alpha (IL-1alpha) for 3 hours and simultaneously conditioned with shear stresses of 0, 0.68, or 13.2 dyne/cm(2) in a parallel-plate flow chamber. In the presence of an inflow buffer containing 100 nmol/L factor X and 10 nmol/L factor VII, production of factor Xa, a measure of functional tissue factor (TF), was determined as the product of outflow concentration of factor Xa (chromogenic assay performed under quasi-static flow conditions after the shear period) and flow rate. Similarly, production of TF pathway inhibitor (TFPI) was estimated as the product of antigenic TFPI (by enzyme-linked immunosorbent assay) in the supernatant and flow rate. In parallel experiments, total RNA was isolated for determination of amplification products of TF mRNA by reverse transcription-polymerase chain reaction. We found that shear stress reduced factor Xa production (mean+/-SE; n=number of experiments) from 13.33+/-1.14 (n=16) fmol/minxcm(2) at 0 shear stress to 5.70+/-2.51 (n=5) and 0.54+/-0.54 (n=4) fmol/minxcm(2) at shear stresses of 0.68 and 13.2 dyne/cm(2), respectively. At the same time, immunogold labeling showed that TF antigen on the endothelial surface increased >5-fold with shear stress, whereas TFPI antigen on the surface increased 2-fold. The secretion of TFPI (appearance of new supernatant TFPI) rose from 7.4+/-2.4 (n=12) x10(-)(3) fmol/minxcm(2) at 0 shear stress to 23.7+/-7.3 (n=9) and 50.2+/-14.3 (n=4) x10(-)(3) fmol/minxcm(2) at 0.68 and 13.2 dyne/cm(2), respectively. TF mRNA amplification products were not markedly changed by shear stress. We conclude that acute application of shear stress reduces functional, but not antigenic, expression of TF by intact, activated endothelial cell monolayers in a manner associated with shear stress-augmented endothelial cell secretion of TFPI.

An in vitro cell culture system to study the influence of external pneumatic compression on endothelial function.

PURPOSE: External pneumatic compression (EPC) is an effective means of prophylaxis against deep venous thrombosis. However, its mechanism remains poorly understood. Understanding of the biological consequences of EPC is an important goal for optimizing performance of the EPC-generating device and providing guidance for clinical use. We present a new in vitro cell culture system (Venous Flow Simulator) that simulates blood flow and vessel collapse conditions during EPC, and we examine the influence of these factors on endothelial cell (EC) fibrinolytic activity and vasomotor function. METHODS: An in vitro cell culture system was designed to replicate the hemodynamic shear stress and vessel wall strain associated with induced blood flow during different modes of EPC. Human umbilical vein endothelial cells were cultured in the system and subjected to intermittent flow, vessel collapse, or a combination of the two. The biologic response was assessed through changes in EC morphology and the expression of fibrinolytic factors tissue plasminogen activator, plasminogen activator inhibitor type 1, profibrinolytic receptor (annexin II), and vasomotor factors endothelial nitric oxide synthase and endothelin-1. RESULTS: The cells remained attached and viable after being subjected to intermittent pulsatile flow (F) and tube compression (C). In F and F + C, cells aligned in the direction of flow after 6 hours. Northern blot analysis of messenger RNA shows that there is an upregulation of tissue plasminogen activator expression (1.95 +/- 0.19 in F and 2.45 +/- 0.46 in FC) and endothelial nitric oxide synthase expression (2.08 +/- 0.25 in F and 2.11 +/- 0.21 in FC). Plasminogen activator inhibitor type 1, annexin II, and endothelin 1 show no significant change under any experimental conditions. The results also show that pulsatile flow, more than vessel compression, influences EC morphology and function. CONCLUSION: Effects on ECs of intermittent flow and vessel collapse, either individually or simultaneously, were simulated with an in vitro system of new design. Initial results show that intermittent flow associated with EPC upregulates EC fibrinolytic potential and influences factors altering vasomotor tone. The system will facilitate future studies of EC function during EPC.

The response of adult human saphenous vein endothelial cells to combined pressurized pulsatile flow and cyclic strain, in vitro.

Adult human saphenous vein endothelial cells (HVEC) were cultured in a compliant tubular device and evaluated by Northern hybridization for the effects of combined pressurized pulsatile flow and cyclic strain on the expression of mRNAs for endothelin-1 (ET-1), endothelial cell nitric oxide synthase (ecNOS), tissue plasminogen activator (tPA), and plasminogen activator inhibitor type 1 (PAI-1). The hemodynamic environment was designed to mimic shear stress conditions at the distal anastomosis of a saphenous vein graft, a common site of intimal proliferation. Steady-state mRNA levels in experimental tubes were expressed relative to that in controls. No changes were observed in ET-1 mRNA after 1 and 24 hr, but a 50% decrease in experimental cultures was observed after 48 hr in the vascular simulating device. Similar results were obtained for ecNOS mRNA, although a subgroup (4 of 11) showed a significant decrease (>50%) by 24 hr. For tPA mRNA, no change was observed after 1 hr, but a significant decrease (>60%) was measured after 24 hr and no message was detectable after 48 hr. Steady-state levels for PAI-1 mRNA remained unchanged through 48 hr of treatment. These results show that pressure, pulsatile flow, and cyclic strain, when applied in concert, differentially alter vasoactive and fibrinolytic functions in HVEC. Moreover, the dramatic decrease in steady-state levels of tPA mRNA is consistent with a shift toward an increased thrombotic state.

Comparison of tissue factor pathway in human umbilical vein and adult saphenous vein endothelial cells: implications for newborn hemostasis and for laboratory models of endothelial cell function.

In this work we have undertaken a comparative study of human umbilical vein endothelial cells (HUVECs) and human saphenous vein endothelial cells (HSVECs) with respect to functional and antigenic tissue factor (TF), tissue factor pathway inhibitor (TFPI), and TF mRNA. Monolayers of each cell type (passage 2, except where specified) were grown to confluence and then activated for 4 h with either 50 U/mL IL-1-alpha or 10 microg/mL tumor necrosis factor-alpha. Activated factor X appearing in supernatant was measured using a chromogenic assay, and both Northern blots and quantitative RT-PCR were performed to assess concentrations of TF mRNA accompanying activation. The role of TFPI was separately determined by ELISA for supernatant TFPI antigen, and by measurements of production of activated factor X in the presence of 0, 5, 15, or 50 microg/mL of an antibody directed against TFPI. To address a non-TF pathway endothelial cell function, antigenic concentrations of tissue plasminogen activator for both cell types was also determined by ELISA. HUVECs were found to produce 2.4- to 3.5-fold more functional TF. No significant HUVEC-HSVEC differences were detected in TF antigen, supernatant TFPI, anti-TFPI affinity for endothelial cell-associated TFPI, TF mRNA or its amplification products, and tissue plasminogen activator. Immunostaining for TF antigen, however, may have failed to detect a modest HUVEC-HSVEC difference. Our finding with respect to functional TF indicates that HUVECs and HSVECs are not equivalent in terms of models for endothelial cell function in small children versus adults.

Stage-specific effects of sonic hedgehog expression in the epidermis.

Sonic hedgehog (Shh) is expressed in the ectoderm of the forming hair follicle and feather bud during normal development. However, inappropriate activation of the Shh signal transduction cascade in human epidermis can cause basal cell carcinoma. Here we show that during normal development of avian skin, Shh is first expressed only after the responsiveness to this protein has been suppressed in most of the surrounding ectodermal cells. Forced expression of Shh in avian skin prior to this time causes a disorganized ectodermal proliferation. However, as skin begins to differentiate, the forced expression of Shh causes feather bud formation. Subsequently, expression of Shh in interfollicular epidermis has little or no morphological effect. Restricted responsiveness to Shh in developing skin has functional consequences for morphogenesis and may have important implications for cutaneous pathologies as well.

Characteristics of vascular wall cells subjected to dynamic cyclic strain and fluid shear conditions in vitro.

We recently developed an in vitro silicone rubber tubular apparatus, the vascular simulating device (VSD), which simulates pressure, flow, and strain characteristics of peripheral arteries (Benbrahim et al., 1994, J. Vasc. Surg. 20, 184-194). In this report, we tested the ability of silicone rubber surfaces to support the growth and differentiation of endothelial cells (EC) and smooth muscle cells (SMC) and studied the effects of arterial levels of pressure, flow, and strain on these properties. Human umbilical and saphenous vein EC and bovine aortic EC and SMC were cultured on coated and uncoated silicone rubber in flat and tubular configurations (6 mm inner diameter) and on tissue culture plastic (TCP). Attachment, growth, and differentiation were compared on these surfaces. In addition, the effects of arterial pressure, flow, and strain conditions on adhesion and subsequent growth and differentiation were studied in the tubular configuration. Attachment and growth of vascular wall cells on fibronectin-coated silicone rubber was similar to that obtained on TCP. Application of arterial levels of pressure, flow, and strain did not alter adhesion of the cells to the tubes. Subsequent passage of these cells demonstrated that attachment, growth, and differentiation (uptake of LDL and expression of factor VIII-related antigen by EC and expression of muscle-specific actin by SMC) were similar in cells derived from experimental and control tubes which were not subjected to arterial conditions. Finally, mRNA expression of specific "housekeeping" genes was similar in cells isolated from experimental and control tubes. We conclude that the VSD supports the culture of viable and differentiated EC and SMC. These experiments demonstrate that it is possible to evaluate the effects of arterial strain and fluid shear on vascular wall cells in vitro, in a configuration similar to the blood vessel wall.

A compliant tubular device to study the influences of wall strain and fluid shear stress on cells of the vascular wall.

PURPOSE: Cellular constituents of the blood vessel wall are continuously subjected, in vivo, to both mechanical and hemodynamic forces, which elicit structural and biologic responses. We have developed a compliant tubular system, the vascular simulating device (VSD), that reproduces these forces, while supporting the attachment and the experimental manipulation of endothelial and smooth muscle cells. METHODS: The VSD consists of a compliant silicone rubber tube coupled to a pump system, which permits the simultaneous application of known levels of pressure and flow, to vascular wall cells cultured on the inner surface of the tube. Seeded cells can be monitored visually under phase contrast or fluorescent optics, as well as harvested and analyzed for biologic responses. RESULTS: The elastic modulus and compliance of the silicone rubber tube are similar to those of canine and human arteries. Endothelial and smooth muscle cells cultured on the lumenal surface of the tubes remain attached and viable after subjecting them to physiologic pulsatile flow and cyclic strain. CONCLUSION: The VSD makes it possible to approximate, in vitro, those forces encountered by vascular wall cells, in vivo and therefore may make it possible to determine whether specific combinations of mechanical and hemodynamic forces are causally associated with specific vascular diseases.

Hemodynamics and aneurysm development in vascular allografts.

PURPOSE: Mechanical and immunologic factors may play a role in the development of native arterial and biologic graft aneurysms. We developed an experimental rat aortic allograft aneurysm model in which segments of infrarenal aorta were transplanted between hypertensive and normotensive rats to study these factors in this model. METHODS: Aortic allografts and autografts were inserted into spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats. Effects of immunologic and antihypertensive therapy were evaluated. Graft diameters were followed up with magnetic resonance imaging and at harvest. Direct-pressure measurements were taken and dp/dtmax (force of ventricular contractions) was calculated before harvest. RESULTS: Autografts remained isodiametric and maintained their histologic architecture. Aneurysmal dilation of transplanted segments occurred in SHR host allografts but not in WKY host allografts. Histologic examination of all allograft specimens noted a rejection reaction characterized by inflammatory cell infiltration and medial smooth muscle cell loss. Antigenic enhancement accelerated aneurysm development in SHR hosts but had no significant effect on WKY hosts. Rates of allograft enlargement and final allograft diameters were similar in antihypertensive treated and untreated SHR hosts. The dp/dtmax in untreated SHR hosts was greatest and differed significantly from that in the WKY rats but only marginally from that in treated SHR hosts. CONCLUSIONS: Immunologic rejection but not abnormal hemodynamics is necessary for development of allograft aneurysm in this model.

Magnetic resonance imaging of small arteries.

Magnetic resonance imaging (MRI) with receive-only surface coil technology was used to visualize and quantitative luminal diameters of small arteries in the rat. MRI measurements of normal and aneurysmal aortas, over a diameter range of 1-3 mm, were closely correlated with direct measurements made visually at laparotomy: measured differences averaged 0.16 mm, and the least-squares regression line (R2 = 0.97, P < 0.001) compared favorably to the line of equivalence, X = Y. This noninvasive but precise imaging modality demonstrates the potential value of using MRI to evaluate the diameter of small vessels, including the postoperative monitoring of arterial bypass graft patency in peripheral regions.

Urokinase activity in the developing avian heart: a spatial and temporal analysis.

Early events in cardiac morphogenesis are characterized by cell migrations and extensive tissue remodeling. This study was undertaken to determine the levels of urokinase in specific regions of the avian heart during early stages of development. Urokinase has previously been shown to be involved in both cell migration and matrix turnover. Elevated urokinase activity and mRNA levels were associated with the onset of ventricular trabeculation and mesenchymal cell migration in the endocardial cushion tissues. Urokinase was localized by immunostaining to the endocardial and mesenchymal cells of the developing atrioventricular canal (AVC) and outflow tract (OFT) as well as with evaginating ventricular endocardium. No immunoreactivity was seen associated directly with the matrix, suggesting that the enzyme remained mostly cell associated, a finding which was confirmed in isolated endocardial cells. Results from this study suggest a role for urokinase in the tissue remodeling and cell migration that occurs during the early stages of cardiac morphogenesis.

Spontaneously hypertensive and Wistar Kyoto rats are genetically disparate.

Spontaneously hypertensive rats (SHR) are one of the most common animal models used to study essential hypertension in humans. Because SHR and normotensive Wistar Kyoto (WKY) rats were both established from the same parental, normotensive Wistar stock, WKY animals have been used almost exclusively as control animals in studies of SHR. Recently, the suitability of WKY rats as normotensive controls for SHR has been challenged. To establish whether or not SHR and WKY rats share the same immunologic backgrounds, we initially performed a series of skin grafting experiments on these animals. In all cases, grafts of SHR donor skin to WKY recipients and of WKY donor skin to SHR recipients resulted in complete rejection within 7 to 10 days. In addition, grafts of WKY donor skin to other WKY recipients resulted in graft rejection. By contrast, skin grafts between SHRs were always accepted. To further characterize the genetic distinctions between SHR and WKY rats, allelic profiles based on a series of immunologic and biochemical markers were established for each strain. These findings clearly establish that SHR and WKY rats differ at the major histocompatibility complex, in specific blood group antigens, and in a panel of isozymic markers. Moreover, whereas SHRs have the same genetic profiles irrespective of source, some colonies of WKY rats are outbred, as judged by their variant allelic profiles.

Increased deposition of basement membrane macromolecules in specific vessels of the spontaneously hypertensive rat.

The genetically determined chronic hypertension manifested in the Spontaneously Hypertensive Rat (SHR) serves as a useful animal model for the study of human essential hypertension. One of the earliest morphologic changes observed in the vasculature of the SHR is the development of a thickened subendothelial space (SES). Neither the biochemical composition nor the anatomic distribution of this early subendothelial deposit has been definitively determined. By combining morphologic, immunologic, and molecular biological approaches, it was demonstrated that by 15 weeks the acellular subendothelial thickening in the vasculature of the SHR results at least in part from the increased synthesis and deposition of basement membrane macromolecules. Moreover, rather than being manifest systemically, this early connective tissue lesion appears to be localized primarily to the aorta and major branches off the aortic arch.

Size-dependent hyaluronate degradation by cultured cells.

Hyaluronate degradation was examined in cultures of vascular wall cells (bovine aortic endothelial cells, rat aortic smooth muscle cells) and in nonvascular cells (chick embryo fibroblasts). The three cell types examined all produced hyaluronidase activity in culture which had a strict acidic pH requirement for activity. This suggested that the enzyme was active only within an acidic intracellular compartment and therefore that hyaluronate degradation occurred at an intracellular site. This was supported by the observation that the presence of hyaluronidase activity alone was not sufficient to ensure degradation of extracellular hyaluronate. Rather, the key limiting factor in this process appeared to be hyaluronate internalization, and this was found to be hyaluronate size-dependent and to a degree, cell-specific. The relationship of these results to morphogenesis and tissue remodeling is discussed.

Isolation of rat aortic endothelial cells by primary explant techniques and their phenotypic modulation by defined substrata.

An efficient and reliable procedure for the isolation and culture of endothelium from large vessels of small animals (e.g., rat) is described, which takes advantage of endothelial cell-extracellular matrix interactions to promote the outgrowth of cells from tissue explants. The procedure may also permit the isolation by nonenzymatic means, of endothelial cells from other vessels and tissues. Rings and opened segments of aortic tissue were placed on a variety of substrates including: untreated tissue culture plastic; films of fibronectin, laminin, type I collagen, and gelatin; gels of type I collagen (Vitrogen, Collagen Corporation, Palo Alto, California), of basement membrane components derived from the EHS sarcoma (Matrigel, Collaborative Research Inc., Lexington, Massachusetts), and of agar and agarose. The medium used was OPTI-MEM or RPMI 1640 (Gibco Laboratories, Grand Island, New York) with 3% or 20% fetal calf serum, and 50 micrograms/ml endothelial cell growth supplement. Only explants on Vitrogen and on Matrigel produced a significant and consistent outgrowth of cells and this occurred shortly after the initiation of explants. Virtually no outgrowth occurred from explants on the other substrata, even after 10 days in culture. On Vitrogen gels, the cells emerged from the explants as single stellate and bipolar cells, whereas those on Matrigel grew as chains and sheets from the edges of the explant. Cells were passaged from both types of gels onto plastic or glass surfaces. The passaged cells isolated from both gel matrices exhibited specific endothelial cell characteristics including a "cobbled" morphology at confluence, positive staining for von Willebrand factor, and uptake of Di-I-Ac-low density lipoprotein. Because rat and other small animal aortic endothelial cells are resistant to isolation by enzymatic treatment, this technique provides a simple means to obtain large numbers of this cell type. Further, the method permits study of endothelial cell functions in vitro, and the roles which the extracellular matrix may play in these processes.

Loss of hyaluronate-dependent coat during myoblast fusion.

Cultured myoblasts were found to exhibit extensive, Streptomyces hyaluronidase-sensitive pericellular coats as revealed by exclusion of particles (fixed red blood cells). These coats are not discernible subsequent to fusion of the myoblasts to form myotubes. The myoblasts contained 2.5 times more hyaluronate attached to their cell surface than myotubes when the data was expressed per unit of protein, but no change in hyaluronate was evident on a per DNA basis. Hyaluronidase activities in the cultures were equivalent when expressed per unit of protein. We conclude that, although the myotubes accumulate larger amounts of protein than myoblasts, there is no compensatory increase in hyaluronate.

Hyaluronate binding and degradation by cultured embryonic chick cardiac cushion and myocardial cells.

Cultured cells obtained from developing chick heart valvular and septal primordial tissues (cardiac cushions) and myocardium were tested for their capacity to bind, internalize, and degrade hyaluronate. A presumptive lysosomal hyaluronidase capable of hyaluronate degradation has been previously isolated and partially characterized from cultures enriched in either cushion tissue cells or myocardial cells (D. H. Bernanke and R. W. Orkin, 1984, Dev. Biol. 106, 351-359). In this study, both types of cultures were found to bind hyaluronate, but only the myocardial cultures could degrade the hyaluronate substrate. The lack of hyaluronate degradative capacity in the mesenchymal cushion tissue cells appears to result from their inability to internalize the macromolecule, thus failing to make it available to the lysosomal hyaluronidase. The data suggest that hyaluronate clearance from the extracellular matrix of the developing cushion is a complex process, involving more than simple extracellular degradation adjacent to the migrating mesenchymal cushion tissue cells. Instead, a sequence of events may be indicated which includes binding of hyaluronate to the cushion tissue cell surfaces and its transport by these cells across the cushion matrix toward the myocardium. The myocardium may be involved in the ultimate removal of hyaluronate from the cardiac jelly.

Hyaluronidase activity in embryonic chick heart muscle and cushion tissue and cells.

Hyaluronidase activity was compared in embryonic chick cardiac cushion and noncushion segments, as well as in cultures of mesenchyme derived from cardiac cushion endocardium (cushion tissue-enriched cultures) and in cultures of myocardial cells at stages critical to heart valve and septum development. Enzyme levels were higher in both heart tissue regions at periods of active cushion tissue mesenchyme migration than after migration ceases, and higher in the cushion region than in the noncushion region at both periods. Hyaluronidase was measured in cells and medium in both types of cultures, with five times greater activity found in the myocardial cultures. The cardiac hyaluronidase from cells and medium of both culture types had an estimated molecular weight of 41,000 to 44,000 and degraded hyaluronate and, to a lesser degree, chondroitin sulfate, at an acidic pH optimum. Ion-exchange chromatography demonstrated that in both culture types, a proportion of the secreted enzyme was more acidic than that found in the cell layer. These studies indicate the potential for hyaluronate degradation by the major cell types present in the developing heart at early stages and that the enzyme responsible is probably a lysosomal enzyme. Therefore, hyaluronate internalization is a likely requirement for degradation, and thus, the turnover of hyaluronate in developing heart valves is more complex than the extracellular degradative process suggested by histochemical data.

Biosynthesis of sulphated macromolecules by rabbit lens epithelium. II. Relationship to basement membrane formation.

Rabbit lens epithelial cells display a similar "cobblestone" morphology and produce the same complement of sulphated macromolecules (also see Heathcote, J.G., and R.W. Orkin, 1984, J. Cell Biol., 99:852-860) whether grown on plastic or glass, dried films of gelatin or type IV collagen with laminin, or on gels of type I collagen. There was no evidence of basement membrane formation by these cells when they were grown on plastic, glass, or dried films. In contrast, cultures that had been grown on gels deposited a discrete basement membrane that followed the contours of the basal surfaces of the cells and in addition, they secreted amorphous basement membrane-like material that diffused into the interstices of the gel and associated with the collagen fibrils of the gel. A significant proportion (approximately 70%) of the heparan sulphate proteoglycan fraction that was secreted into the culture medium (fraction MI) when the cells were grown on plastic became associated with the cell-gel layer in the gel cultures. Further, when basement membrane was isolated by detergent extraction, greater than 90% of the 35S-labeled material present was in this heparan sulphate proteoglycan.