Suspensions Of Smooth Muscle Cells Research Articles

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods.2-5 Herein, we present a method to rapidly self-assemble cells into 3D tissue rings that can be used in vitro to model vascular tissues.To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring.6,7 These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength8 which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (<30 kPa).9,10 Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function.

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods.2-5 Herein, we present a method to rapidly self-assemble cells into 3D tissue rings that can be used in vitro to model vascular tissues. To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring.6,7 These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength8 which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (<30 kPa).9,10 Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function.

Differences in the Requirements for Cryopreservation of Porcine Aortic Smooth Muscle and Endothelial Cells

One of the basic requirements for the production of tissue-engineered constructs is an effective means of storing both the constructs and the cells that will be used to make them. This paper reports on the cryopreservation of porcine aortic smooth muscle and endothelial cells intended for the production of model vascular constructs. We first determined the cell volume, nonosmotic volume, and the permeability parameters for water and the cryoprotectant dimethyl sulfoxide (Me(2)SO) in these cells at 2-4 degrees and 22 degrees C. The following results were obtained: Table unavailable in HTML format. Using a cell culture assay, both cell types were shown to tolerate threefold changes in cell volume, in either direction, without significant injury. Although these data suggested that single-step methods for the introduction and removal of 10% w/w Me(2)SO should be effective, an additional mannitol dilution step was adopted in order to reduce the time required for removal of the Me(2)SO. Following cooling at 0.3, 1, or 10 degrees C/min and storage at less than -160 degrees C, the survival of porcine aortic smooth muscle cell suspensions, measured by a cell culture assay, was inversely related to cooling rate; at 0.3 degrees C/min, recovery was >80%. The survival rate for aortic endothelial cells was directly related to cooling rate over the range tested and was >80% at 10 degrees C/min.

Estimating oxygen transport resistance of the microvascular wall.

The problem of diffusion of O(2) across the endothelial surface in precapillary vessels and its utilization in the vascular wall remains unresolved. To establish a relationship between precapillary release of O(2) and vascular wall consumption, we estimated the intravascular flux of O(2) on the basis of published in vivo measurements. To interpret the data, we utilized a diffusion model of the vascular wall and computed possible physiological ranges for O(2) consumption. We found that many flux values were not consistent with the diffusion model. We estimated the mitochondrial-based maximum O(2) consumption of the vascular wall (M(mt)) and a possible contribution to O(2) consumption of nitric oxide production by endothelial cells (M(NO)). Many values of O(2) consumption predicted from the diffusion model exceeded M(mt) + M(NO). In contrast, reported values of O(2) consumption for endothelial and smooth muscle cell suspensions and vascular strips in vitro do not exceed M(mt). We conjecture that most of the reported values of intravascular O(2) flux are overestimated, and the likely source is in the experimental estimates of convective O(2) transport at upstream and downstream points of unbranched vascular segments.

Transformed rat arterial smooth muscle cells induce platelet aggregation

Atherogenesis is characterized by a proliferation of arterial smooth muscle cells that may be of transformed nature. Platelets are implicated in the progression of atherosclerotic lesions through thrombosic complications. The present study was designed to investigate whether transformed arterial smooth muscle cells (SMC) could specifically aggregate platelets. We used rat transformed arterial SMC lines, V6- and V8-lines, that we had previously established. Experiments were performed with an in vitro homologous rat system. Suspensions of SMC were added without any other aggregating agent to rat heparinized platelet-rich plasma (PRP) in a coagulo-aggregometer. The effect of transformed V6-line and V8-line SMC was compared to that of their normal parental counterparts, V6- and V8-parent cells. Suspensions of transformed SMC induced, in a dose-dependent manner, an immediate and reversible ADP-like platelet aggregation. The amplitude of platelet aggregation was much higher with addition of transformed cells than of the corresponding control SMC (7.39 ± 0.75 cm vs. 0.85 ± 0.62 cm with 2 × 10 6 SMC, V6-line vs. V6-parent cells, respectively). ADP-like aggregation did not significantly differ between the two transformed V6- and V8-lines. ADP-like platelet aggregation was also obtained with supernatants of transformed SMC suspensions, the amplitude being higher with supernatants than with cell suspensions (21.0 ± 3.64 cm vs. 6.8 ± 1.22 cm with 1.0 × 10 6 V8-line cells, supernatant vs. cell suspension, respectively). The transformed SMC-induced aggregation of platelets was inhibited by apyrase (125 μM) and iodoacetate (25mM) and thus was ascribable to ADP released by the SMC. In addition, all suspensions of SMC, normal or transformed, but not their supernatants, induced plasma clotting after variable coagulation times. Coagulation was inhibited by hirudin (25 to 100 U/ml) and phospholipase A2 (10 U/ml) indicating thrombin generation through activity of the SMC membrane tissue factor. The present results show that transformed arterial smooth muscle cells may directly aggregate platelets via a release of ADP and this could be of pathophysiological relevance for thrombosis associated with atherosclerosis.

Spatial changes of [Ca2+]i and contraction caused by phorbol esters in vascular smooth muscle cells.

In A7r5 smooth muscle cell suspensions, 12-deoxyphorbol 13-isobutyrate (DPB) and phorbol 12,13-dibutyrate (PDB), active phorbol esters for protein kinase C (PKC), increased the intracellular Ca2+ concentration ([Ca2+]i), but 4 alpha-PDB, an inactive phorbol ester, did not. Digital images of the fura 2 fluorescence from single cells revealed that DPB caused elevation of Ca2+ in localized peripheral regions, followed by expansion of this elevated Ca2+ level throughout the cytoplasm. High K+ also induced a dynamic change of [Ca2+]i. DPB and high K+ increased the wrinkles and distortions in the flexible growth surface beneath the cells. In Ca(2+)-depleted media, DPB did not affect [Ca2+]i but substantially increased wrinkles. The increase could be eliminated by staurosporine, a specific inhibitor of PKC. DPB increased the particulate PKC activity in a concentration-dependent manner. These results suggest that PKC activation induces cellular contractions through two distinct mechanisms, one dependent on and another independent of the [Ca2+]i increase.

Source of activator calcium in isolated guinea pig and human gastric muscle cells

The source of Ca2+ responsible for contraction was examined in suspensions of smooth muscle cells and in perfused single muscle cells from guinea pig and human stomach. In both preparations removal of Ca2+ from the medium or addition of the Ca2+ channel blocker methoxyverapamil had no effect on the contractile response to various agonists, including cholecystokinin octapeptide (CCK-8) and acetylcholine, but inhibited the response to high extracellular K+ by 76-82%. Repeated stimulation of guinea pig or human single muscle cells in Ca2+-free medium, or in the presence of methoxyverapamil caused a progressive decrease and eventual abolition of contractile response; response was restored on restitution of Ca2+ to the medium or elimination of methoxyverapamil. Measurement of 45Ca2+ content in guinea pig muscle cells showed that CCK-8 had no effect on the rate of Ca2+ influx but increased the rate of Ca2+ efflux transiently by sixfold. Net peak efflux coincided with the time of peak contraction and was stoichiometrically related to the degree of contraction. Equipotent, maximally effective contractile doses of CCK-8, acetylcholine, and methionine-enkephalin caused equivalent degrees of net Ca2+ efflux. The results indicate that contractile agonists cause release of Ca2+ from a depletable intracellular store in gastric muscle cells. The release is accompanied by a dose-dependent increase in Ca2+ efflux and is capable of sustaining an initial maximal contraction. Repeated contractile activity requires influx of Ca2+ from extracellular sources.

Evidence for two cellular sodium compartments in suspensions of smooth muscle cells.

Unidirectional transmembrane fluxes of 24Na were examined in suspensions of enzymatically dispersed toad stomach muscle cells. Compartmental analysis of 24Na influx indicated that 24Na exchange consisted of two kinetically distinguishable components in these cells: a large pool (approximately 45 mmol/l cell H2O) with a rapid rate of exchange [half time (t1/2) approximately 5.8 min] and a smaller pool (approximately 26 mmol/l cell H2O) that exchanged more slowly (t1/2 approximately 69 min). Only the latter appeared ouabain sensitive; thus we postulated that the rate of exchange of this pool might be determined by the inward leak of Na+. Based on this assumption, we derived estimates for Na+ influx rate (0.9 X 10(-12) mol . cm-2 . s-1) and Na+ permeability (0.3 X 10(-8) cm/s), which presumably represent upper limits for these parameters in smooth muscle. The location and significance of the rapidly exchanging pool were not resolved. The exchange of both cellular Na+ pools was increased by the ionophore monensin. Thus we speculated that the rapidly exchanging pool was associated with a small population of permeable (high Na+) smooth muscle cells, while the slowly exchanging pool was associated with smooth muscle cells with normal Na+ permeability (low-Na+ cells).

Beta-adrenergic stimulation of 42K influx in isolated smooth muscle cells.

The present studies were designed to examine the effect of the beta-adrenergic agent isoproterenol (ISO) on 42K influx into isolated smooth muscle cells. Unidirectional 42K influx studies were carried out as described previously using suspensions of smooth muscle cells derived from the toad stomach. Results from these studies indicated that ISO (10(-4) M) produces an approximate threefold increase in 42K influx within 1 min after drug addition. This response is transient such that the flux rate returns to near basal levels within 5 min after drug addition. The stimulatory effect of ISO on 42K influx is blocked by pretreatment with ouabain (1-7.5 X 10(-4) M). ISO thus appears to increase 42K influx by stimulating the Na+-K+ pump in the smooth muscle cells. Other findings indicate that the stimulatory effect of ISO on Na+-K+ pumping is dose dependent, blocked by the beta-antagonist pindolol, and mimicked by dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP). These findings support our hypothesis that ISO elicits a beta-adrenergic, cAMP-dependent activation of the Na+-K+ pump in isolated smooth muscle cells.

Preparation of smooth muscle cell suspensions from the rabbit oviduct and prostaglandin binding analysis.

The preparation, viability, and prostaglandin (PG) binding characteristics of dispersed smooth muscle cells from the rabbit oviduct have been determined. Cell suspensions were prepared by enzymatic degradation of the intracellular matrix and subsequent mechanical dispersion with a wide bore pipette. The digestion media consisted of a modified Hanks' Balanced Salt Solution, pH 7.1, containing 1.6 U/mg wet wt elastase and 8 U/mg wet wt collagenase. This method provided a yield of single smooth muscle cells of approximately 3 x 10(6) cells/100 mg wet wt within 3-4 h of organ removal. Cell viability was determined by trypan blue dye exclusion, retention of lactate dehydrogenase, absence of 57Co-EDTA uptake, and ouabain sensitivity of cationic transport. Roughly 80% of the isolated cells remained viable after the digestion procedure. The dispersed cells specifically bound [3H]PGE2 and [3H]PGF2 alpha. Scatchard analysis of the binding data revealed separate homogenous populations of high affinity sites for both PGE2 and PGF2 alpha. The equilibrium dissociation constants and total sites per cell were 0.55 nM and 11,332 for PGE2 and 0.19 nM and 5,154 for PGF2 alpha, respectively. Specific labeled PG binding was inhibited in a concentration-dependent manner by increasing amounts of unlabeled PG. Inhibition of labeled PGE2 binding by unlabeled PGF2 alpha, and vice versa were negligible, except at high concentrations. The results indicate that smooth muscle cells can be enzymatically dispersed from the rabbit oviduct with minimal damage. Also, these cells possess distinct specific binding sites for PGE2 and PGF2 alpha that differ in regard to affinity and total number of sites per cell.