Endothelial regeneration. V. Inhibition of endothelial regrowth in arteries of rat and rabbit

Endothelium was removed from the carotid artery of both rat and rabbit using a 2F balloon catheter. The regrowth of endothelium progressed from either end of the vessels but had stopped after 2 weeks in the rabbit and 6 weeks in the rat. Total outgrowth along the vessel was 3 mm in the rabbit and approximately 10 mm in the rat, and by 12 weeks a large zone of the vessel was still devoid of endothelium. Autoradiographic data showed that 12 weeks after the injury endothelial cell replication was reduced to background values. When these vessels were reinjured with a nylon catheter, endothelial cell replication was initiated and the wound rehealed, but these cells did not progress further into the denuded region. Morphologic examination of arteries 12 weeks after balloon denudation showed that endothelial cells, abutting the luminal smooth muscle cells, were larger and not aligned with flow. The luminal smooth muscle cells formed a loose junction with the leading edge of the endothelium and exhibited an extracellular material on their luminal aspect. These results demonstrate that endothelial regeneration will stop before cellular regrowth is complete and that cell senescence is not responsible. The presence of smooth muscle cells on the luminal surface appears to be related to this inhibition of growth.     

Interaction of acetylcholine and cholecystokinin with dispersed smooth muscle cells.

Isolated gastric smooth muscle cells were prepared from the stomach of Bufo marinus by successive incubation in collagenase without added trypsin. Contraction was determined by image-splitting micrometry and expressed as the mean percentage decrease in cell length from control. Peak contractile response was attained within 30 s. Dose-response curves constructed from peak responses showed that the maximal responses to CCK-OP (37.2 +/- 3.8%), acetylcholine (35.3 +/- 2.5%), and Ca2+ (42.3 +/- 0.9%) were similar. The D50s for octapeptide of cholecystokinin (CCK-OP) and acetylcholine were around 10(-12) M and 10(-11) M, respectively. The response to a combination of submaximal concentrations of acetylcholine and CCK-OP exceeded the individual responses but did not exceed the maximal response to either agent alone. A low concentration of atropine (5 X 10(-10) M) inhibited specifically the maximal response to acetylcholine. A high concentration of atropine (5 X 10(-8) M) inhibited partially the maximal response to CCK-OP but had no effect on the maximal response to Ca2+. It was concluded that 1) dispersed gastric smooth muscle cells are highly sensitive to stimulation; 2) CCK-OP has a direct (myogenic) contractile effect on gastric smooth muscle; and 3) the effect of CCK-OP and acetylcholine are mediated by separate receptors.     

Angiogenesis-like activity of endothelial cells co-cultured with VEGF-producing smooth muscle cells

A number of strategies have been investigated to improve therapeutic vascularization of ischemic and bioengineered tissues. In these studies, we genetically modified vascular smooth muscle cells (VSMC) to promote endothelial cell proliferation, migration, and formation of microvascular networks. VSMCs were virally transduced to produce vascular endothelial growth factor (VEGF), which acts as a chemoattractant and mitogen of endothelial cells (EC). VSMCs transduced with VEGF(165) cDNA produced significant levels of the protein (2-4 ng/10(5) cell/day). The proliferation of ECs increased after exposure to VEGF-transfected SMCs or their conditioned media. The chemotactic response of ECs to the VEGF-producing cells was explored in two in vitro systems, the modified Boyden chamber assay and a 2-D fence-style migration assay, and both demonstrated increased migration of ECs in response to VEGF-transfected cells. Furthermore, endothelial cells seeded on top of the VEGF-transfected SMCs formed capillary-like structures. These results suggest that VSMCs genetically modified to produce VEGF could be a potential delivery mechanism to enhance endothelial cell migration and subsequent capillary formation, which in turn could improve vascularization of ischemic or regenerating tissue. Furthermore, this system could potentially be used as an in vitro test bed for evaluation of novel angiogenic and anti-angiogenic compounds.     

Interaction of endothelial and smooth muscle cells of the pig aorta in primary culture

In primary coculture of endothelial and smooth muscle cells, the muscle cells preserve their specialized phenotype (high myosin content) contrary to a pure smooth muscle cell culture. The addition of thrombin to the coculture-system results in loss of specialized phenotype of smooth muscle cell i.e. the relative myosin content decreases.     

Migration of human vascular endothelial and smooth muscle cells.

Migration of endothelial and smooth muscle cells was studied in vitro by measuring the increase in surface area at specific time intervals of confluent cell colonies advancing under agarose gels that contained both Morgan's medium 199 and variuos types of sera. First passage cultures of endothelial cells or 3 to 6 passage smooth muscle cells were plated into wells punched in agarose gels, at a seeding density of 50,000 cells per well. At zero time the size of the cell colonies was 35.4 sq. mm. +/- standard error 0.1. Irradiation (1500 rads) did not affect the expansion of the cell colonies although 3H-thymidine uptake was inhibited. Endothelial cells migrated under the agarose gels concentrically as contiguous sheets. When exposed to either 20 per cent platelet-poor plasma serum, platelet-rich plasma serum, or whole blood serum, the average increase in surface area was approximately 9 sq. mm. per day. In contrast, arterial smooth muscle cell colonies expanded with an increment of approximately 9 sq. mm. per day when exposed to 10 per cent platelet-poor plasma serum but 12 sq. mm. per day when exposed to 10 per cent platelet-rich plasma serum (p less than 0.001). Platelet factors also had stimulatory effects on the migration of venous smooth muscle cells. Cytochalasin B, dibutyryl cyclic AMP, and theophylline inhibited the migration of both endothelial and smooth muscle cells, but the latter responded more to the inhibitory effects of all three agents. It is concluded that in contrast to vascular smooth muscle, endothelial cells do not require platelet factors for migration and are less responsive to specific inhibitors affecting cell movement.     

Endothelial regeneration. II. Restitution of endothelial continuity

With a modified balloon catheter the endothelium of rat thoracic aortae was completely removed to study the interaction between two important responses of the vessel wall to intimal injury: endothelial regeneration and formation of an intimal fibrocellular thickening. Endothelial cells deriving from the uninjured intercostal arteries regenerated by migration followed by proliferation and proceeded as a continuous sheet at a rate of approximately 0.07 mm. per day in the circumferential direction and approximately 6 times faster in the axial direction. Smooth muscle cells appeared in the intima only in areas which were not covered by regenerating endothelium 7 days after injury. The smooth muscle cells formed a multilayered fibrocellular intimal lesion which reached the maximal thickness after 3 weeks. The continuous sheet of regenerating endothelium covered the intimal smooth muscle cells at a slower rate; 6 weeks after injury large areas located most distant from the source of regenerating endothelium still showed modified smooth muscle cells lining the lumen. However, platelets did not adhere to these smooth muscle cells, and the total amount of intimal thickening did not increase between 3 and 6 weeks after injury. We conclude that, in response to intimal injury, endothelial regeneration precedes the accumulation of intimal smooth muscle cells, and that injured intimal areas, which are rapidly covered by continuous endothelium, are protected from the development of a fibrocellular intimal lesion.     

Interaction of foamy cells from experimental hyperlipemia rabbits with smooth muscle cells and the endothelial surface of intima preparation]

In the development of atherosclerosis (AS), circulating monocytes emigrate into and accumulate in the intima as foamy cells. Interaction of the lipid laden macrophage (M phi) with cells of the arterial wall may contribute to the formation of atheromatous plaque. Using subcutaneous and peritoneal foamy cells (FC) collected from diet-induced hyperlipemia rabbits, the authors observed the influence of lipid laden on macrophage's functions relevant to AS lesion formation. In comparison with normal M phi, peritoneal FC were 4.8 and 5.4 fold more adhesive to the endothelial surface of intima preparation and the smooth muscle cells (SMC), while the adhesion rate produced by subcutaneous FC were increased 0.79 and 0.16 fold. SMC migration stimulated by both FC-conditioned medium slowed 5.78 and 5.90 fold increased respectively as in comparing with the control's, whereas normal M phi-conditioned medium only gave a 2.3 fold increase of SMC migration stimulation. In addition, both FC-conditioned medium stimulated SMC growth making 1.96 and 2.59 fold increase, and normal M phi-conditioned medium produced a 1.3 fold increase as compared with the control's. The results suggest that lipid-laden macrophages, may accelerate AS development due to changes of their biological properties. Since more than 95% of peritoneal macrophages as well as macrophages in the pleura, pericardial and synovial cavities from experimental hyperlipidemia rabbits are fully filled with lipid assuming the morphologic characteristics of atheromatous intimal foamy cells, it is considered that these cells will be valuable as the model.     

Aortic smooth muscle cells express and secrete vascular endothelial growth factor.

We examined whether cultured bovine aortic smooth muscle (ASM) cells express VEGF. RNA blot analysis of total cellular RNA derived from ASM cells demonstrates the expression of the VEGF gene. ASM cells release in the medium a VEGF-like endothelial cell mitogen which binds to heparin-sepharose and has an apparent molecular weight of 40-45 kDa as assessed by an HPLC gel filtration column. Consistent with VEGF, this mitogen does not stimulate the proliferation of ASM cells. Immunoblot analysis of the bioactive material with an antibody specific for VEGF demonstrates the presence of a major immunoreactive band with an apparent molecular mass of 23 kDa and a minor band with a molecular mass of approximately 18 kDa, in reducing conditions. The major band has very similar apparent molecular weight as the 165 amino-acid species of human recombinant VEGF of folliculo-stellate cells derived VEGF. These data demonstrate the expression and synthesis of VEGF by cultured ASM cells and suggest that the 164 amino-acid species is the predominant molecular form of the growth factor secreted by such cells. VEGF released by ASM cells may play a paracrine role in the maintenance of the integrity of the endothelial lining or in the abnormal proliferation of the vasa vasorum which takes place in atherosclerosis.     

Interaction of collagen and smooth muscle cells in aortic biomechanics

To elucidate the contribution of smooth muscle to the mechanical properties of artery and to study its interaction with other tissue components, an enzymolysis technique has been used. Fresh canine artery was cut into circumferential strips, and a stressrelaxation test was performed in a Krebs-Ringer solution while continuously bubbling an O₂−CO₂ gas mixture. An aortic smooth muscle stimulant, norepinephrine, was perfused during the stress-relaxation test, before and after removing collagen with collagenase. The collagen-free artery did not show any contraction due to the smooth muscle, in contrast to the intact artery. This indicates that there may be important interactions between the collagen matrix and smooth muscle. This new technique can be used to study the contribution of smooth muscle to the mechanical properties of aorta.     

Pregnancy is associated with hypotrophy of carotid artery endothelial and smooth muscle cells

It is known that blood flow through the carotid artery is decreased during pregnancy, which may be due to a pregnancy-associated increase in the sensitivity of this artery to vasoconstrictors. Recent studies have shown that alteration of blood flow or pressure could remodel some arteries over a short time frame. However, the possibility of remodelling of the carotid artery during pregnancy has not yet been examined. Therefore, the aim of the present study was to study the morphometrical and stereological characteristics of guinea-pig carotid artery during different stages of pregnancy (non-pregnant, early- pregnant, mid-pregnant, late-pregnant, n = 8-10 for each group). The cross-sectional area of the different layers of the carotid artery and the cross-sectional area of endothelial and smooth muscle cells were measured using both light and electron microscopy. The values of internal diameter and cross-sectional area of adventitia were not significantly different, regardless of the pregnancy status. In contrast, external diameter, wall thickness and cross-sectional areas of media and intima progressively and significantly decreased during pregnancy. In addition, volume/surface density ratio of intima and media also significantly and progressively decreased during pregnancy, suggesting hypotrophy of endothelial and smooth muscle cells of carotid artery. Indeed, electron microscopy revealed that the size, defined as cross-sectional area, of endothelial and smooth muscle cells was significantly decreased during different stages of pregnancy. It is concluded that during pregnancy there is thinning of the intimal and medial layers of guinea-pig carotid artery, which reflect pregnancy- associated hypotrophy of carotid artery endothelial and smooth muscle cells.      

Early proto-oncogene expression in rat aortic smooth muscle cells following endothelial removal.

To study the mechanism(s) of vascular smooth muscle cell proliferation in vivo, mRNA levels of c-fos, c-jun, and c-myc were determined by Northern blot analysis following vascular balloon de-endothelialization (BDE). Medial smooth muscle cells (SMC) were separated and studied by enzymatic digestion of the vessel wall. mRNA levels of c-fos and c-jun from aortic smooth muscle cells (SMC) were simultaneously induced within 30 minutes of BDE and declined to baseline by 1.5 hours, c-myc mRNA did not begin to increase until 1 hour after vascular injury. Levels of c-myc peaked at 2 hours and were sustained for an additional 4 hours before gradually declining. Smooth muscle cells derived from enzyme-treated control aortae that did not undergo BDE expressed c-fos and c-jun, but showed no evidence of c-myc message. In contrast, nonenzymatically treated, non-BDE whole aortae (containing both media and adventitia) demonstrated a prominent c-myc signal, but failed to express c-fos and c-jun. Corresponding examination of adventitia derived from enzyme-treated aortae showed this tissue to be a source of all three proto-oncogenes. The results of this study demonstrate the earliest in vivo molecular markers of vascular injury reported to date and implicate SMC proto-oncogene expression in the initiation of SMC proliferation. Furthermore these findings suggest two avenues for proto-oncogene induction, that are due to (1) vessel wall manipulation and (2) humoral stimulation.     

Rat myometrial smooth muscle cells express endothelial nitric oxide synthase

We examined if rat myometrial cells in culture generate nitric oxide (NO) and express various isoforms of NO synthase (NOS). Myometrial cells isolated from rats on day 18 of gestation were incubated with various stimulators and inhibitors of NOS for 24 and 48 h, and NO production was evaluated by measuring nitrites in the media and NOS proteins in the cell lysates. NO was produced by myometrial cells and its production inhibited by N(G)-methyl-L-arginine (L-NMMA). This inhibition was reversed by L-arginine (3 mM). Interleukin-1beta (IL- 1beta) significantly stimulated NO production, in a dose-dependent manner. The IL-1beta-stimulated NO production was inhibited by the NOS inhibitor, L-NMMA, whose effects were reversed by L-arginine. Abundant NOS III protein was detectable in freshly isolated myometrial cells, and this was maintained in culture in the presence of fetal bovine serum (FBS; 10%). In the absence of FBS, NOS III levels decreased significantly (by 90%) within 24 h. In contrast, NOS I and NOS II proteins were undetectable in freshly isolated muscle cells and in cells cultured without IL-1beta. However, NOS II protein in these cells was induced by IL-1beta. Thus, NO is produced by myometrial cells through the NOS III isoform, and the myometrial NO may be important in maintaining uterine quiescence during pregnancy.      

Human aortic smooth muscle cells promote arteriole formation by coengrafted endothelial cells

Collagen-fibronectin gels containing Bcl-2-transduced human umbilical vein endothelial cells (Bcl-2-HUVEC) implanted in the abdominal walls of immunodeficient mice form mature microvessels invested by host-derived smooth muscle cells (SMC) by 8 weeks. We tested the hypothesis that coengraftment of human aortic SMC (HASMC) could accelerate vessel maturation. To prevent SMC-mediated gel contraction, we polymerized the gel within a nonwoven poly(glycolic acid) (PGA) scaffold. Implanted grafts were evaluated at 15, 30, and 60 days. Acellular PGA-supported protein gels elicited a macrophage-rich foreign body reaction and transient host angiogenic response. When transplanted alone, HASMC tightly associated with the fibers of the scaffold and incorporated into the walls of angiogenic mouse microvessels, preventing their regression. When transplanted alone in PGA-supported gels, Bcl-2-HUVEC retained the ability to form microvessels invested by mouse SMC. Interestingly, grafts containing both Bcl-2-HUVEC and HASMC displayed greater numbers of smooth muscle alpha-actin-expressing cells associated with human EC-lined arteriole-like microvessels at all times examined and showed a significant increase in the number of larger caliber microvessels at 60 days. We conclude that SMC coengraftment can accelerate vessel development by EC and promote arteriolization. This strategy of EC-SMC coengraftment in PGA-supported protein gels may have broader application for perfusing bioengineered tissues.     

剪切力条件下血管内皮细胞与平滑肌细胞的相互作用

血管内皮细胞(endothelial cells,ECs)与平滑肌细胞(smooth muscle cells,SMCs)是血管壁的主要细胞,它们之间的相互作用在维持血管正常的生理功能以及心血管疾病的发生发展过程中至关重要。为真实模拟ECs与SMCs体内条件下的位置关系与生长状态,人们建立了多种共培养系统。介绍当前几种常用的能够加载流动剪切力的共培养系统,并分别比较其优势与不足;简要总结剪切力条件下ECs与SMCs相互作用对ECs与SMCs表型与分布、SMCs生长与迁移、ECs表面相关黏附分子表达的影响。研究表明,一氧化氮(NO)、细胞因子、microRNA等可以作为信号分子介导ECs与SMCs之间的相互作用。     

Clusters of proliferating endothelial cells and smooth muscle cells in rabbit carotid arteries

Schwarz and Benditt found clustering of replicating cells in aortic endothelium in 1976 and discussed how homeostasis of the arterial wall is maintained through this nonrandom distribution of replicating cells. However, it is still unclear how cells of vascular walls turnover. In order to address this issue, we evaluated distribution of the cells in mitotic cycle, labeled by Ki67‐immunostaining, in serial histological sections of twelve carotid arteries of six adult male Japanese rabbits. As a result, a total of 1713 Ki67‐positive endothelial cells (ECs) and 1247 Ki67‐positive smooth muscle cells (SMCs) were identified. The Ki67‐positivity rate in ECs and SMCs were about 0.048% and 0.0027%, respectively. Many of the Ki67‐positive cells clustered in two (EC, 37%; SMC, 33%), three to four (EC, 8%; SMC, 28%), and five to eight cells (EC, 5%; SMC, 10%). Clusters having more than eight cells were not found. Thus, it can be speculated that the cell division of proliferating ECs and SMCs occur four times at most. These novel findings offer great insights for better understanding of the mechanism that underlies cell number regulation of the blood vessel.     

Electrical coupling between smooth muscle cells and endothelial cells in pig coronary arteries

 The control of smooth muscle cells by endothelial cells has been well established by the identification of vasoactive factors released by the endothelial cells. In contrast, the possibility that smooth muscle cells influence the endothelial cells has been considered rarely. Some results suggest possible electrical communication between the smooth muscle and the endothelial cells but proof is lacking. We therefore tested for electrotonic conduction of signals from smooth muscle cells to endothelial cells. The endothelium was removed from half of a strip of porcine coronary artery. In a partitioned chamber, rectangular hyperpolarization or depolarization was applied to the de-endothelialized region by field stimulation. The resulting membrane potential changes in the smooth muscle cells spread electrotonically along the media into the area with intact endothelium. We recorded from endothelial cells to determine whether this electrical signal spreads into endothelial cells. Hyperpolarization or depolarization initiated in smooth muscle cells was recorded consistently in endothelial cells. This demonstrates a functional electrotonic propagation from smooth muscle to endothelial cells. Received: 23 May 1996 / Received after revision: 3 September 1996 / Accepted 16 September 1996     

Chlamydia pneumoniae facilitates monocyte adhesion to endothelial and smooth muscle cells

Chlamydia pneumoniae has been linked to atherosclerotic heart disease. However, there is a limited knowledge by which C. pneumoniae gain access to atheromatous lesions. The adhesion of C. pneumoniae -infected circulatory component(s) to endothelium and smooth muscle cells represents the first step in an inflammatory response. We examined the ability of viable as well as heat inactivated C. pneumoniae to infect human monocytes and subsequently the ability of infected monocytes to adhere to human coronary artery endothelial cells (HCAEC) and human coronary smooth muscle cells (HCSMC). Our results demonstrate susceptibility of monocytes to in vitro chlamydial infection. Inclusions of varying sizes and intensities were observed 3-5 days after inoculation with viable C. pneumoniae. Monocytes infected with heat inactivated organisms revealed no inclusions, in keeping with the observations of uninfected monocytes. Moreover, monocytes infected with viable C. pneumoniae adhered preferentially to HCAEC and HCSMC, as compared to uninfected monocytes or monocytes harbouring heat inactivated Chlamydia.     

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.