改进构建后的全生物化组织工程血管的组织结构与生物力学特性

目的改进体外构建的生物化组织工程血管(TEBV),研究其生物力学特性,获得能承载生理血流力学作用的血管替代物。方法用酶消化法制备猪颈总动脉脱细胞支架,加压灌注结合散点注射种植犬胸主动脉平滑肌细胞(VSMC),用自制的血管自动旋转流体培养系统培养3周,随后再用加压灌注法种植犬胸主动脉内皮细胞(EC),继续培养至4周,取材做HE染色。光镜和电镜观察VSMC及EC在支架内的生长情况。用力学测试仪器检测TEBV的应力-应变关系、拉伸弹性回复率以及最大断裂强度和长度。结果在血管自动旋转流体培养系统,VSMC种植3周在中膜层已经大量均匀分布,EC种植7天后已形成连续完整的EC单层;而应用单独的加压灌注法VSMC种植3周时仍不能均匀分布,EC种植1周后仍分布不均匀,未能形成连续的EC单层。TEBV培养至第4周,中膜层有大量VSMC生长,内膜层则形成连续的EC单层;透射电镜下可见VSMC、EC亚微结构与生理状态的细胞结构相似,可见缝隙连接等细胞连接方式,VSMC可产生新的胶原蛋白。扫描电镜可见EC在支架上生长良好,细胞轮廓清晰,形成连续的单层。力学特性检测结果显示:TEBV的粘弹性、拉伸弹性回复率以及最大断裂强度均接近生理血管。结论血管自动旋转流体培养系统在脱细胞血管支架上联合种植VSMC和EC,有利于种子细胞在支架腔面和管壁内均匀生长,改进了TEBV的组织结构。改进构建的TEBV生物力学特性接近生理血管,将有利于TEBV更好地承载血流动力学的作用。     

Fourier transform infrared spectroscopy application to vascular biology: comparative analysis of human internal mammary artery and saphenous vein wall

Saphenous vein (SV) and internal mammary artery (IMA) are used for aorto-coronary bypass grafting. IMA is considered to be the graft of choice for coronary revascularization having a long-term patency compared to SV. The aim of this study is to investigate the structure of vascular wall using a new technical approach. We analysed the chemical composition of vessel wall layers (total lipid, lipid ester and protein) of 25 vascular segments (19 SV and 6 IMA) using Fourier transform infrared spectroscopy (FTIR). FTIR analysis showed that in intima layer lipid ester and protein concentration (expressed as arbitrary units) was significantly higher in SV (lipid ester = 0.020 +/- 0.002; protein = 0.449 +/- 0.022) than in IMA (lipid ester = 0.014 +/- 0.002; protein = 0.342 +/- 0.032). Moreover, the percentage of lipid ester on total lipid was significantly higher in SV (intima = 54.7 +/- 2.9%; media = 78.4 +/- 4.9%; adventitia = 83.9 +/- 8.3%) wall layers compared to IMA ones (intima = 37.3 +/- 4.9%; media = 45.4 +/- 3.8; adventitia = 57.1 +/- 4.8). These data suggest that a different chemical composition of wall layers could also be responsible for the morphological modifications observed in SV after grafting.     

The effects of botulinum toxin A and papaverine on human saphenous vein and internal mammary artery grafts: an in vitro study

IntroductionAutologous saphenous vein (SV) and internal mammary artery (IMA) are used as bypass conduits during coronary artery bypass graft surgery. Vasospasm of the arterial and venous grafts may constitute a significant clinical problem. Pretreatment with a vasodilator drug of the graft ex vivo or intraluminal injection before implantation may be used for spasm prophylaxis. This in vitro study was designed to assess the vasoactive effects and time-dependent changes of botulinum toxin A (BTX-A) and papaverine pretreatment on vasospasm of human SV and IMA grafts. Also, histomorphology of the vessels was assessed.Material and methodsSV and IMA segments were suspended in organ baths, and isometric contraction responses to 2 different concentrations of 5-hydroxytryptamine (5-HT) and endothelin-1 (ET-1) were recorded after incubation with 2 different concentrations of BTX-A and papaverine at 2 time points (0 h and 2 h).ResultsThe results revealed the following: 1) incubation with BTX-A and papaverine relaxes both SV and IMA rings contracted with 5-HT and ET-1; 2) the duration of the relaxant effect of BTX-A lasts longer than papaverine; and 3) no apparent histomorphological changes were observed in the grafts under light microscopy.ConclusionsThis study demonstrates that in human SV and IMA grafts, pretreatment with both BTX-A and papaverine are safe and have a potent inhibitory effect depending on the vessel and vasoconstrictor agent. The long-lasting vasodilatory effect of BTX-A on vascular smooth muscle may provide promising results in the prevention of venous and arterial graft spasm.     

Mechanical stretching of human saphenous vein grafts induces expression and activation of matrix-degrading enzymes associated with vascular tissue injury and repair.

After coronary artery bypass surgery, saphenous vein graft occlusion occurs through tissue remodeling. Although a likely trigger, the role of preparative mechanical injury incurred by the graft is not yet understood. We studied the early effects of simple mechanical injury on human saphenous vein grafts by exposing them to longitudinal stretch, a deformation which potentially occurs during surgery. We then maintained ex vivo for up to 7 days matched pairs of experimentally stretched and nonstretched (control) vein segments and examined the expression and activation of matrix metalloproteinases (MMPs) and integrin alphav, molecules implicated in vascular remodeling. At peak expression on day 3, stretched vein secreted 177 +/- 16% active MMP-2 (P < 0.01), 161 +/- 36% (P < 0.05) pro-MMP-9, and contained 206 +/- 18% (P < 0.01) alphav, a receptor for active MMP-2, compared to control. In situ gelatinase activity was present in the intima and adventitia of stretched veins, but not of control, and correlated spatially with expression of alphav. Stretch also increased severalfold cell proliferation (1.27 +/- 0.4 vs. 0.23 +/- 0.05% in control, P < 0.05), as assessed by bromodeoxyuridine incorporation. Furthermore, we found that cell proliferation colocalized with gelatinase activity and alphav in the adventitia. Our results show that a single longitudinal stretch of vein grafts produces significant changes in the expression and activation of key molecules in vascular remodeling. We also found support for the notion that the adventitial layer contributes to vein graft remodeling.     

Mechanical and biochemical properties of human cervical tissue

The mechanical integrity of cervical tissue is crucial for maintaining a healthy gestation. Altered tissue biochemistry can cause drastic changes in the mechanical properties of the cervix and contribute to premature cervical dilation and delivery. We present an investigation of the mechanical and biochemical properties of cervical samples from human hysterectomy specimens. Three clinical cases were investigated: nonpregnant hysterectomy patients with previous vaginal deliveries; nonpregnant hysterectomy patients with no previous vaginal deliveries; and pregnant hysterectomy patients at time of cesarean section. Tissue samples were tested in confined compression, unconfined compression and tension. Cervical tissue samples for the three clinical cases were also subjected to biochemical analysis. Biochemical assays measured cervical tissue hydration, collagen content, collagen extractability and sulfated glycosaminoglycan (GAG) content. Results from the mechanical tests indicate that cervical stroma has a nonlinear, time-dependent stress response with varying degrees of conditioning and hysteresis depending on its obstetric background. It was found that the nonpregnant tissue was significantly stiffer than the pregnant tissue in both tension and compression. Further, collagen extractability, sulfated GAG content and hydration were substantially higher in the pregnant tissue. This study is the first important step towards the attainment of an improved understanding of the complex interplay between the molecular structure of cervical tissue and its macroscopic mechanical properties.     

Co-expression of elastin and collagen leads to highly compliant engineered blood vessels

Elastin synthesis and physiologic compliance are significant challenges in blood vessel tissue engineering. Here, we report that a biocompatible elastomeric scaffold can support the co-expression of elastin and collagen, which likely yielded the physiologic compliance in the constructs. A biodegradable elastomer, poly(glycerol sebacate), was fabricated into highly porous tubular scaffolds. Primary baboon arterial smooth muscle cells (SMCs) were seeded in the lumen of the scaffolds followed by a 1-week culture under gentle perfusion. Circulating endothelial progenitor cells (EPCs) isolated from baboon peripheral blood was seeded directly on the smooth muscle layer in the lumen on day 8. The constructs were perfused using a pulsatile flow system for another 2 weeks before characterization. In another set of experiments, the SMCs were cultured for 7 weeks and were co-cultured for 1 week with the EPCs. Constructs obtained using either set of culture conditions contained elastin and collagen: Masson's trichrome stain showed a circumferential collagen band in the constructs, and elastin was evident from its characteristic autofluorescence, Verhoff's stain, and amino acid analysis of insoluble remnants after hot alkali digestion. All constructs had a confluent cellular lumen with cells well-dispersed throughout the scaffolds. At physiologic pressures, the compliance of the 8-week construct was comparable to human arteries as observed in pressure-diameter testing. Combination of elastomeric scaffolds, co-culture of EPC and SMC, and mechanical conditioning appears to encourage the expression of a more natural extracellular matrix and lead to physiologically-relevant compliance; both are major challenges in blood vessel tissue engineering.     

Diabetes and reactivity of isolated human saphenous vein

Helical strips of saphenous veins from diabetic (n = 8) and non-diabetic (n = 18) humans were studied in vivo for their responsiveness to several vasoactive agents. Following application of passive force (approximately 20.0 mN), venous strips from non-diabetic humans often developed spontaneous phasic contractile activity (12 out of 18 patients; 2-5 contractions/min). These intrinsic changes in force were seen in venous strips from only one diabetic patient. The phasic contractions were not altered by treatment with phentolamine, whereas the calcium channel blocker, D-600, and calcium-free solution (1.0 mM EGTA) inhibited the phasic contractions. Saphenous veins from diabetic patients developed less maximal, active tension in response to norepinephrine than those from non-diabetic patients. Contractile responses to serotonin, angiotensin II, and elevated potassium concentration in saphenous veins from diabetic patients were not different from those in veins from non-diabetic patients. These observations demonstrate attenuated development of active tension in response to alpha-adrenergic receptor activation and reduced spontaneous contractile activity in venous smooth muscle from diabetic patients.     

以踝管区动脉穿支为蒂隐神经-大隐静脉营养血管皮瓣的显微解剖

目的:为踝管区动脉穿支隐神经-大隐静脉营养血管皮瓣设计提供解剖学依据。方法:30侧经动脉内灌注红色乳胶成人下肢标本,解剖观测踝管区动脉穿支、胫后动脉肌间隙支及邻近动脉吻合。结果:踝管区近侧筋膜穿支2支,外径(0.8±0.3)mm(0.4~1.3mm),中侧筋膜穿支0~2支,外径(0.8±0.2)mm(0.4~1.2mm),远侧筋膜穿支2~3支,外径(1.0±0.4)mm(0.5~2.0mm),共同构成踝管区浅深筋膜及皮肤营养血管。在小腿内侧下1/3段,胫后动脉肌间隙支2~3支,平均外径(1.1±0.3)mm(0.5~2.5mm),骨皮穿支1~2支,平均外径(1.0±0.3)mm(0.4~2.0mm)。上述穿支形成隐神经、大隐静脉营养血管及深、浅筋膜血管网。结论:踝管区筋膜穿支支数较多,与胫后动脉肌间隙支吻合丰富,以踝管区筋膜穿支为蒂的隐神经-大隐静脉营养血管皮瓣,旋转点在内踝平面,可用于转位修复足前部的软组织缺损。     

大隐静脉剥脱术误扎股动、静脉的治疗体会

目的 探讨大隐静脉剥脱中高位结扎大隐静脉时误扎股静脉或/和股动脉的治疗方法及其临床效果。方法 回顾性分析 2011 年9月—2015 年10月徐州仁慈医院收治外院转诊的7例大隐静脉剥脱术中高位结扎大隐静脉时误扎股静脉或/和股动脉患者的临床资料,其中男4例、女3例,年龄39～61岁;股静脉破裂1例,股静脉切断结扎4例,股动脉切断结扎1例,股动、静脉切断结扎1例。7例患者中,股静脉破裂修补1例,屈髋屈膝位吻合血管3例,对侧大隐静脉移植桥接修复2例,人工血管移植桥接修复1例。结果 本组7例患者术后血管造影示术侧股动、静脉通畅。术后随访6个月~ 3年,所有患者患肢功能满意,行走正常。结论 实施大隐静脉高位结扎手术时,应熟练掌握解剖基础;术中误扎股动、静脉后,应根据缺损程度及时采取适当的术式进行修复重建,可获得满意疗效。     

Biomechanics of the saphenous artery and vein in spontaneous hypertension in rats

Incremental compliance and distensibility of certain muscular arteries were recently reported to be normal or slightly increased in hypertension at the same pressure levels. In this work biomechanical properties of isolated perfused and superfused veins and large muscular arteries from saphenous bed from male spontaneously hypertensive (SHR) and Wistar–Kyoto (WKY) rats were compared in vitro. Outer diameter of cylindrical vessel segments was measured and intraluminal pressure (IP) was changed cyclically. We found larger contractile response to methoxamine (1.06×10⁻⁵ mol/l) in SHR arteries compared to WKY (active strain e.g. at 100 mmHg IP: 7.12±4.1 vs 0.35±0.46%). Resting incremental distensibility was higher (e.g. 100 mmHg IP: 3.4±0.4×10⁻⁶ vs 1.2±0.3×10⁻⁷ m²/N), elastic modulus lower (e.g. 100 mmHg IP: 3.7±0.6×10⁵ vs 27±7.6×10⁵ N/m²) in the arteries from SHR in pressure range of 60–110 mmHg. After papaverine administration (2.8×10⁻⁴ mol/l) the artery became more rigid, thus the increased incremental elasticity of SHR artery might be due to the enhanced smooth muscle tone. However, compared at in vivo pressure levels the differences were negligible suggesting a shift in the elastic parameters toward the higher operation pressures. Saphenous vein of SHR had larger diameter, than that of WKY, while in the wall thicknesses no difference were found (therefore external radius-wall thickness ratio was larger, e.g. at 6 mmHg IP: 15.9±3.0 vs 8.1±0.7). Consequently, lumen capacity of the vein was also higher in SHR, however, elastic parameters did not exhibit significant differences. We conclude that pressure-distensibility curve of muscular type arteries like SHR saphenous artery is shifted to higher pressure levels compared with that of normotensive controls. This shift is due to the enhanced smooth muscle contractility. The unchanged elasticity of veins suggests that the arterial deformations in SHR are not primary but secondary alterations.     

Responses to potential vasoactive substances of isolated mammary blood vessels from lactating sows

The purpose of the investigation was to examine the response of the milk vein and the mammary artery to potential vasoactive substances in lactating sows. The response on artery and vein segments from the same sow was measured for the following substances: noradrenaline (NA), serotonin (5-HT), prostaglandin F2alpha (PGF2alpha), prostacyclin (PGI2), histamine (Hi) and potassium (K+). Interestingly, the contractile force in the mammary vein segments expressed per weight unit (mN/mg) was 1.5-2.5 fold larger than in the artery segments when NA, 5-HT and PGF2alpha were used, but similar for K+, Hi and PGI2. In vein segments, the order of sensitivity to the substances expressed by their pD2 values was 5-HT > NA = PGF2alpha > Hi > K+. The present findings suggest that NA, 5-HT, PGF2alpha, PGI2, and Hi are involved in mammary blood flow regulation in the sow, and the mammary venous system may be as important as the mammary arterial system in this regulation.     

Functionality, growth and accelerated aging of tissue engineered living autologous vascular grafts

Living autologous tissue engineered vascular-grafts (TEVGs) with growth-capacity may overcome the limitations of contemporary artificial-prostheses. However, the multi-step in vitro production of TEVGs requires extensive ex vivo cell-manipulations with unknown effects on functionality and quality of TEVGs due to an accelerated biological age of the cells. Here, the impact of biological cell-age and tissue-remodeling capacity of TEVGs in relation to their clinical long-term functionality are investigated. TEVGs were implanted as pulmonary-artery (PA) replacements in juvenile sheep and followed for up to 240 weeks (～4.5years). Telomere length and telomerase activity were compared amongst TEVGs and adjacent native tissue. Telomerase-activity of in vitro expanded autologous vascular-cells prior to seeding was <5% as compared to a leukemic cell line, indicating biological-aging associated with decreasing telomere-length with each cellular-doubling. Up to 100 weeks, the cells in the TEVGs had consistently shorter telomeres compared to the native counterpart, whereas no significant differences were detectable at 240 weeks. Computed tomography (CT) analysis demonstrated physiological wall-pressures, shear-stresses, and flow-pattern comparable to the native PA. There were no signs of degeneration detectable and continuous native-analogous growth was confirmed by vessel-volumetry. TEVGs exhibit a higher biological age compared to their native counterparts. However, despite of this tissue engineering technology related accelerated biological-aging, growth-capacity and long-term functionality was not compromised. To the contrary, extensive in-vivo remodeling processes with substantial endogenous cellular turnover appears to result in "TEVG rejuvenation" and excellent clinical performance. As these large-animal results can be extrapolated to approximately 20 human years, this study suggests long-term clinical-safety of cardiovascular in vitro tissue engineering and may contribute to safety-criteria as to first-in-man clinical-trials.     

Adherence of human saphenous vein endothelial cell monolayers to tissue-engineered biomatrix vascular conduits

This study investigates the adherence and retention under in vitro flow conditions of endothelium grown on the luminal surface of 4-mm-internal-diameter, biomatrix vascular conduits. The biomatrix vascular conduits are produced in living animals and consist of a naturally formed extracellular matrix wall incorporating a polyester mesh. We propose that the microarchitecture of the luminal surface may be conducive to endothelial cell (EC) seeding and to the formation of a firmly adherent endothelium without prior treatment of the surface with cell adhesives. ECs were isolated from segments of human saphenous vein and grown to confluence in a culture. Cultured cells were characterized by morphology and immunocytochemistry with anti-CD31, Von Willebrand factor, smooth muscle actin, cytokeratin, and the lectin Ulex Europaeus agglutinin. After culture, ECs were seeded (1 x 10(6) cell/mL) by rotation onto the luminal surface of 20-cm-long, biomatrix vascular conduits (n = 3). The seeded conduits were incubated for 72 h, and at set time points postseeding (1, 24, 48, and 72 h), the morphology, percentage luminal surface cover, and cell density (cell/cm(2)) were determined from en face preparations and histological cross sections. After 72 h in culture, the seeded conduits were subjected to a nonpulsatile flow for 1 h with culture media. A flow rate of 480 mL/min generated physiological-range shear stresses of 12 dyn/cm(2) on the endothelialized surface. After the flow, the conduits were fixed for histology, and the EC morphology and percentage luminal surface cover were determined. Quantification of the extent of luminal surface endothelialization, preflow and postflow, and cell densities at confluence was performed with digital imaging light microscopy and image analysis software. An analysis of the results demonstrated that confluent EC monolayers may be established on the luminal surface of biomatrix vascular conduits within 48 h. The formed endothelium was firmly adherent and was retained under physiological-range flow.     

A collagen-based scaffold for a tissue engineered human cornea: physical and physiological properties

Stabilized collagen-glycosaminoglycan scaffolds for tissue engineered human corneas were characterized. Hydrated matrices were constructed by blending type I collagen with chondroitin sulphates (CS), with glutaraldehyde crosslinking. A corneal keratocyte cell line was added to the scaffolds with or without corneal epithelial and endothelial cells. Constructs were grown with or without ascorbic acid. Wound-healing was evaluated in chemical-treated constructs. Native, noncrosslinked gels were soft with limited longevity. Crosslinking strengthened the matrix yet permitted cell growth. CS addition increased transparency. Keratocytes grown within the matrix had higher frequencies of K+ channel expression than keratocytes grown on plastic. Ascorbic acid increased uncrosslinked matrix degradation in the presence of keratocytes, while it enhanced keratocyte growth and endogenous collagen synthesis in crosslinked matrices. Wounded constructs showed recovery from exposure to chemical irritants. In conclusion, this study demonstrates that our engineered, stabilized matrix is well-suited to function as an in vitro corneal stroma.     

A comparative study on the mechanical properties of the healthy and varicose human saphenous vein under uniaxial loading

Abstract

Saphenous Vein (SV) due to fatness, age, inactiveness, etc. can be afflicted with varicose. The main reason of the varicose vein is believed to be related to the leg muscle pump which is unable to return the blood to the heart in contradiction of the effect of gravity. As a result of the varicose vein, both the structure and mechanical properties of the vein wall would alter. However, so far there is a lack of knowledge on the mechanical properties of the varicose vein. In this study, a comparative study was carried out to measure the elastic and hyperelastic mechanical properties of the healthy and varicose SVs. Healthy and varicose SVs were removed at autopsy and surgery from seven individuals and then axial tensile load was applied to them up to the failure point. In order to investigate the mechanical behaviour of the vein, this study was benefitted from three different stress definitions, such as 2nd Piola-Kichhoff, engineering and true stresses and four different strain definitions, i.e. Almansi-Hamel, Green-St. Venant, engineering and true strains, to determine the linear mechanical properties of the SVs. A Digital Image Correlation (DIC) technique was used to measure the true strain of the vein walls during load bearing. The non-linear mechanical behaviour of the SVs was also computationally evaluated via the Mooney-Rivlin material model. The true/Cauchy stress–strain diagram exhibited the elastic modulus of the varicose SVs as 45.11% lower than that of the healthy ones. Furthermore, by variation of the stress a significant alteration on the maximum stress of the healthy SVs was observed, but then not for the varicose veins. Additionally, the highest stresses of 4.99 and 0.65 MPa were observed for the healthy and varicose SVs, respectively. These results indicate a weakness in the mechanical strength of the SV when it becomes varicose, owing to the degradation of the elastin and collagen content of the SV. The Mooney-Rivlin hyperelastic and the Finite Element (FE) data were finally well compared to the experimental data.     

The caliber of the human long saphenous vein and its congenital variations

The purpose of the present study is to evaluate the caliber of the normal human long saphenous vein (LSV) in order to verify the occurrence of congenital narrowings. The LSV morphology was evaluated by the dissection of 32 cadaveric limbs, and by ultrasonography of 102 healthy living subjects. The LSV caliber was constant in most of the limbs, showing only a mild and progressive increase from the ankle to the groin. Furthermore, great individual variation in LSV caliber was found. A segmental narrowing of the LSV was present in 39.8% of limbs. The narrow segment was visible with the naked eye during dissection or by ultrasonography in 22.4% of cases (LSV hypoplasia). In the remaining 17.4% the caliber was so reduced that it could only be detected microscopically (LSV aplasia). In relation to the narrow segments, the main ascending flow was shunted in a collateral vein running within the superficial hypodermis. The narrow segments of the LSV had a weaker and less muscular wall than did those of normal caliber. Hypoplasia and aplasia of the LSV are probably due to segmental failure in the development of the vessel, and represent a risk factor for varicosis. In fact, the ascending flow is shunted from the LSV in a collateral vein that runs in the yielding superficial fatty layer of the hypodermis. Furthermore, the high incidence of LSV segmental hypoplasia and aplasia has also to be considered whenever this vein is used as an arterial graft, because of the marked anatomical remodelling.     

原位静脉动脉化与静脉动脉间置后静脉的实验形态学研究

目的：探讨原位静脉动脉化与静脉动脉间置后，静脉的组织学和超微结构变化。在１８只犬后肢设计原位大隐静脉动脉化，静脉动脉间置实验模型，对原位大隐静脉动脉化和静脉动脉间置后不同时间（２、４、８、１６ｗ）静脉管壁的变化，进行了实验形态学观察。结果：①原位静脉动脉化后早期内皮细胞损伤较轻微，中、晚期主要表现为管腔内皮细胞损伤较轻微，管腔的扩张和中膜平滑肌的增生，肥厚；②静脉动脉间置早期内皮细胞有广泛脱落，中膜平滑肌细胞肿胀，细胞间积液，中晚期变化主要为内皮不规则增生，肥厚，中膜平滑肌不同程度的增生与纤维化，使血管腔呈现不同程度的狭窄。结论：原位静脉动脉化后静脉呈现结构上的“动脉化”倾向，并且较静脉动脉间置更有利于保持血管的通畅。     

轴向扭转作用下血管的力学特性和组织重建

血管由于身体活动或经历手术而经常承受轴向扭转(或扭曲)作用,然而有关血管在扭转作用下的研究却很少。本文首先总结血管经受扭转的临床症状,然后描述扭转作用下血管的力学特性以及本构模型;继而进一步讨论最新的有关扭转作用下血管重建的研究,包括生物体外器官培养实验、活体动物实验、数学模型的分析和模拟。希望这篇综述将促进今后对血管在扭转作用下力学特性和重建更多、更深入的研究。     

A method to isolate morphologically distinct clones of smooth muscle cells from human saphenous vein

The monoclonal theory of atherosclerosis postulates that a certain subpopulation of vascular smooth muscle cells (VSMC) is selectively expanded in response to pathological stimuli thereby contributing to the formation of atherosclerotic plaques. VSMC cloning experiments will be important in characterizing the phenotypic composition of VSMC in atherosclerotic plaques. However, the difficulty in cloning human VSMC is well recognized. Here a technique is described that produced multiple clones from human saphenous vein. The clones could be divided into two categories based on their distinctly different morphology: (1) spindle-shaped; and, (2) epithelioid-shaped. Each clone expressed smooth muscle-a-actin and calponin, two smooth muscle-specific differentiation markers. The clonal study presented here reports for the first time that phenotypically heterogeneous smooth muscle cells coexist within human saphenous veins.