Combining cell sheet technology and electrospun scaffolding for engineered tubular,aligned, and contractile blood vessels

Herein we combine cell sheet technology and electrospun scaffolding to rapidly generate circumferentially aligned tubular constructs of human aortic smooth muscles cells with contractile gene expression for use as tissue engineered blood vessel media. Smooth muscle cells cultured on micropatterned and N-isopropylacrylamide-grafted (pNIPAm) polydimethylsiloxane (PDMS), a small portion of which was covered by aligned electrospun scaffolding, resulted in a single sheet of unidirectionally aligned cells. Upon cooling to room temperature, the scaffold, its adherent cells, and the remaining cell sheet detached and were collected on a mandrel to generating tubular constructs with circumferentially aligned smooth muscle cells which possess contractile gene expression and a single layer of electrospun scaffold as an analogue to a small diameter blood vessel's internal elastic lamina (IEL). This method improves cell sheet handling, results in rapid circumferential alignment of smooth muscle cells which immediately express contractile genes, and introduction of an analogue to small diameter blood vessel IEL.     

Circumferentially aligned fibers guided functional neoartery regeneration in vivo

An ideal vascular graft should have the ability to guide the regeneration of neovessels with structure and function similar to those of the native blood vessels. Regeneration of vascular smooth muscle cells (VSMCs) with circumferential orientation within the grafts is crucial for functional vascular reconstruction in vivo. To date, designing and fabricating a vascular graft with well-defined geometric cues to facilitate simultaneously VSMCs infiltration and their circumferential alignment remains a great challenge and scarcely reported in vivo. Thus, we have designed a bi-layered vascular graft, of which the internal layer is composed of circumferentially aligned microfibers prepared by wet-spinning and an external layer composed of random nanofibers prepared by electrospinning. While the internal circumferentially aligned microfibers provide topographic guidance for in vivo regeneration of circumferentially aligned VSMCs, the external random nanofibers can offer enhanced mechanical property and prevent bleeding during and after graft implantation. VSMCs infiltration and alignment within the scaffold was then evaluated in vitro and in vivo. Our results demonstrated that the circumferentially oriented VSMCs and longitudinally aligned ECs were successfully regenerated in vivo after the bi-layered vascular grafts were implanted in rat abdominal aorta. No formation of thrombosis or intimal hyperplasia was observed up to 3 month post implantation. Further, the regenerated neoartery exhibited contraction and relaxation property in response to vasoactive agents. This new strategy may bring cell-free small diameter vascular grafts closer to clinical application.     

Circumferential alignment of vascular smooth muscle cells in a circular microfluidic channel

The circumferential alignment of human aortic smooth muscle cells (HASMCs) in an orthogonally micropatterned circular microfluidic channel is reported to form an in vivo-like smooth muscle cell layer. To construct a biomimetic smooth muscle cell layer which is aligned perpendicular to the axis of blood vessel, a half-circular polydimethylsiloxane (PDMS) microchannel is first fabricated by soft lithography using a convex PDMS mold. Then, the orthogonally microwrinkle patterns are generated inside the half-circular microchannel by a strain responsive wrinkling method. During the UV treatment on a PDMS substrate with uniaxial 40% stretch and a subsequent strain releasing step, the microwrinkle patterns perpendicular to the axial direction of the circular microchannel are generated, which can guide the circumferential alignment of HASMCs during cultivation. The analysis of orientation angle, shape index, and contractile protein marker expression indicates that the cultured HASMCs reveal the in vivo-like cell phenotype. Finally, a fully circular microchannel is produced by bonding two half-circular microchannels, and the HASMCs are cultured circumferentially inside the channels with high alignment and viability for 5 days. These results demonstrated the creation of an in vivo-like 3D smooth muscle cell layer in the circular microfluidic channel which can provide a bioassay platforms for in-depth study of HASMC biology and vascular function.     

Structural relationships between the endothelial actin system and the underlying elastic layer in the distal interlobular artery of the rat kidney

Summary The structure of the intracellular actin filaments and the extracellular matrices was studied in the distal interlobular arteries in the rat kidney, employing three different morphological techniques, including rhodamin-phalloidin staining of cryosections, resorcin-fuchsin staining of paraffin sections, and a cold dehydration procedure for electron microscopy. The endothelial cells possess longitudinally running stress fibers. The inner elastic layer is composed of meshworks of elastic fibers encompassing numerous pores. The smooth muscle cells containing abundant actin filaments are arranged circumferentially around the vascular axis. The endothelial stress fibers are found mainly in the basal half of the endothelial cells, and anchor onto the basal cell membranes. The elastic meshworks send off longitudinal branch fibers to contact the endothelial cell membranes at the anchoring sites of stress fibers. In addition circumferential branches run toward the smooth muscle cells. The functional significance of the intracellular contractile apparatus and the extracellular tensile component in small arteries was discussed.     

Eliminating Arterial Pulsatile Strain by External Banding Induces Medial but Not Neointimal Atrophy and Apoptosis in the Rabbit

We have examined the role of vessel pulsation and wall tension on remodeling and intimal proliferation in the rabbit infrarenal abdominal aorta. A rigid perivascular polyethylene cuff was used to reduce vessel systolic diameter by 25%, producing a region of reduced circumferential strain. At 6 weeks postoperatively, reduced circumferential strain caused medial atrophy, with 45% reduction of medial area and 30% loss of medial smooth muscle cells. Apoptotic cell death was indicated by DNA fragmentation, propidium iodide staining, and cell morphology. Cuffing the aorta after balloon denudation produced medial atrophy but did not inhibit neointimal growth. At 1 week postoperatively, intimal thickness was slightly decreased in regions with reduced strain; however, intimal thickening in regions of reduced strain was not different from control segments at 3 weeks postoperatively (intimal area was 0.37 ± 0.05 mm² with reduced strain and 0.50 ± 0.08 for controls, mean ± SEM). We conclude that circumferential strain is a major factor controlling medial structure and cell number, whereas growth of the neointima after injury is not significantly affected by either reduced strain or extensive medial cell death. Vessel cuffing represents a new model of blood vessel remodeling in vivo that involves extensive smooth muscle cell apoptosis.     

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.     

Novel insights into the elastic and muscular components of the human trachea

Despite its probable importance in health and disease, the elastic tissue in the trachea has rarely been investigated. In addition, various aspects of the trachealis muscle are controversial. The aim of this study was to clarify this clinically relevant anatomy. Ten cadaveric tracheobronchial specimens (age range 68–101 years; seven males; no major airway pathology) were qualitatively investigated by microdissection. Serial histologic sections from multiple sites in three specimens were analyzed after staining for elastin. Findings were correlated with observations from video tracheobronchoscopies. An extensive and prominent meshwork of elastic tissue was found within the trachea and bronchi. Elastic fibers were predominantly longitudinal and aggregated into discrete bundles within the membranous wall of the trachea and main bronchi; a discrete fibroelastic membrane bridging the membranous wall of the trachea; and vertical laminae connecting the ends of successive cartilages. The longitudinal elastic bundles continued into the segmental bronchi, becoming thinner and more circumferentially distributed. Trachealis consisted of a transverse layer of smooth muscle deep to the fibroelastic membrane of the membranous wall of the trachea, together with scattered longitudinal muscle bundles, mostly embedded within the fibroelastic membrane in the distal half of the trachea. In conclusion, there is an extensive but relatively neglected elastic framework within the tracheobronchial tree. This is likely to have major clinical relevance to the pathophysiology of respiratory disease and ageing. The trachealis muscle is more complex than previously stated. Clin. Anat. 22:689–697, 2009. © 2009 Wiley‐Liss, Inc.     

Quantification of Bladder Smooth Muscle Orientation in Normal and Spinal Cord Injured Rats

Spinal cord injuries (SCI) often lead to severe bladder dysfunctions. Our previous studies have demonstrated that following SCI, rat bladder wall tissue became hypertrophied, significantly more compliant, and changed its mechanical behavior from orthotropic to isotropic. In order to elucidate the link between the tissue microstructure and mechanical properties of the wall, we have developed a novel semi-automated image analysis method to quantify smooth muscle bundle orientation and mass fraction in the bladder wall tissues from normal and 10 day-post-SCI rats. Results of the present study revealed that there were significant (p < 0.05) increases in smooth muscle area fractions as well as significantly (p < 0.001) fewer cell nuclei per muscle area in the SCI groups compared to the normal groups. Furthermore, while the normal rat bladders exhibited predominant smooth muscle orientation only in the longitudinal direction, the SCI rat bladders exhibited smooth muscles oriented in both the circumferential and longitudinal directions. These results provide first evidence that bladder smooth muscle cells exhibit hypertrophy rather than hyperplasia and developed a second, orthogonal orientation of smooth muscle bundles following SCI. The results of the present study corroborate our previous mechanical anisotropy data and provide the basis for development of structure-based constitutive models for urinary bladder wall tissue.     

Histopathologic phenomena at the site of percutaneous transluminal coronary angioplasty: the problem of restenosis

Seventeen postangioplasty cases were morphologically studied at postmortem. Four of the eleven, early and intermediate cases (few hours to 1 month from angioplasty to death), revealed intraluminal thrombi, although in only two cases were those thrombi occlusive. Almost all of the nine early cases (eight of nine) exhibited intimal disruptions. Except for two of these cases in which circumferential and/or longitudinal dissections were present, the remainder of the intimal cracks were superficial and of limited extent. Limited dissection between intima and media is not considered a serious or detrimental local event. The early cases showed an aneurysmal dilatation of the plaque-free segment of the arterial wall in eccentric plaques. This finding was interpreted as the result of uneven distribution of the dilating force (circumferential stress) on the aterial wall. Late cases (survival over 1 month) revealed characteristic medial and intimal lesions indicative of the initial dilatation injury. It is hypothesized that intrinsic arterial wall changes (medial disruption) at the plaque-free segment and the resulting altered arterial geometry at the site of dilatation have a significant hemodynamic effect on the vascular conduit and may enhance and sustain the myoproliferative intimal response.     

Comparison of biomechanical and histological properties in dog carotid arteries injured by neointima or intimal thickening

A general formula (Oka and Azuma's equation) has been rigorously derived for the circumferential wall tension in a hollow cylindrical tube in equilibrium. To evaluate the validity and usefulness of Oka and Azuma's equation, T = P(1) x r(1) - P(2) x r(2) (T, circumferential wall tension; P(1) and P(2), internal and external pressures of the tube; r(1) and r(2), the corresponding internal and external radii), we experimentally investigated changes in circumferential wall tension of noninjured (control) and injured dog common carotid arteries by using a newly developed apparatus with a photo- and X-ray-sensitive image sensor. We also studied histological features of the control and injured arteries with special reference to the relation of biomechanical properties. Two types of animal models with injured arteries--balloon-induced neointima or external collar-induced intimal thickening--were adopted in the present study. In the control arteries, the circumferential wall tension was experimentally confirmed to change from negative to positive by an increase in intraluminal pressure ranging from 50 to 180 mmHg. The critical intraluminal pressure that produced 0 dyne/cm of the circumferential wall tension was around 135 mmHg. The activation of arterial smooth muscles caused a significant increase in the critical pressure in the control arteries. In the arteries injured by neointima, the critical intraluminal pressure was significantly lower than that in the control. The activation of smooth muscles also significantly increased the critical pressure in the injured arteries. Histological examination demonstrated the existence of a circumferential neointimal formation along with a shortening of the internal diameter. In other arteries injured by intimal thickening, the circumferential wall tension was always negative at intraluminal pressure ranging from 50 to 180 mmHg. Newly developed structures consisted of elastic and collagen fibers, smooth muscles, and extracellular matrix in the intima and media of the injured arteries. These experimental findings suggest that the circumferential wall tension of dog common carotid arteries has been confirmed experimentally to become negative. We have also concluded that circumferential wall tension calculated with Oka-Azuma's equation may be one of the best parameters for evaluating changes in the biomechanical and histological properties of pathologically injured arteries.     

具有周向微槽结构管状血管支架制备及诱导平滑肌细胞的取向生长

背景：对小口径血管组织工程化而言,平滑肌细胞的周向排列要求彻底改变以前支架的简单多孔结构,代之以能够诱导血管平滑肌细胞三维周向取向和排列新型微观结构。 目的：观察微槽结构对平滑肌细胞体外定向诱导的影响。 方法：用静电纺丝、熔融纺丝并利用溶剂/非溶剂和热压的方式制得了具有两层管壁、外壁具有周向微沟槽结构的仿生管状血管支架,用胶原蛋白固定改性后,在其上种植人脐静脉血管平滑肌细胞。扫描电镜和荧光显微镜观察支架不同缠绕角度对平滑肌细胞定向诱导能力的影响。 结果与结论：①选择比例为5∶95的氯仿/乙醇溶液,浸润时间为5 s,可以使乳酸-羟基乙酸共聚物电纺纤维和乳 酸-ε-己内酯共聚物熔纺纤维之间形成很好的粘连,形成支架。②通过碱降解使支架表面含有羧基,以1-(二甲基胺丙基)-3-乙基碳化二亚胺为缩合剂在支架表面接枝胶原。X射线光电子能谱证实了支架表面胶原大分子的存在。③当纤维之间的编织角度为30°即网孔尺寸适当时,细胞能在支架内部和表面大面积生长。④具有两层管壁结构的仿生管状血管支架具有良好的细胞相容性,其表面周向微槽结构对平滑肌细胞的取向排列具有明显的诱导作用。提示在电纺层外面再熔纺缠绕降解聚合物是制备管状仿生血管支架的可行方法。血管平滑肌细胞能沿着纤维暨微沟槽方向一致取向排列。     

The initiation of intimal thickening in human arteries

To obtain more detailed information about the relationship of intimal thickening to defects in the elastin structure of the arterial wall, the internal elastic lamina and subsequent elastin formation in the intima was studied by very oblique sectioning of paraffin sections of the arterial wall, and by scanning electron microscopy of formic acid digested preparations. Comparison was made in the same subject between the internal thoracic artery (a vessel showing only slight intimal thickening) and the anterior descending coronary (which usually develops advanced intimal thickening). There was no evidence of penetration of the normal fenestrations of the internal elastic lamina by medial smooth muscle cells. This took place through major defects of this lamina and resulted in a change from transverse to longitudinal orientation of these cells and the accompanying elastin fibers of the intima. A further condensation of elastin (greater for the internal thoracic) occurred in the intima subjacent to the endothelial cells.     

Location of stretch receptors in the trachea and bronchi of the dog.

1. Using single fibre vagal afferent recording techniques, we have investigated the longitudinal and circumferential location of slowly adapting stretch receptors in a functionally isolated, in situ segment of the trachea in dogs. 2. We have also studied the depth of these receptors within the airway wall and their response to reflex and drug induced contraction of the airway smooth muscle. 3. Thirty-four per cent of the receptors studied were in the extrathoracic trachea; calculations indicate that about 17-1% of all airway stretch receptors lie outside the thorax. 4. All the receptors were located in the membranous posterior wall of the trachea, and those tested responded more to transverse than to longitudinal stretching of the wall. 5. Receptors in the extrathoracic trachea continued to function after the regional mucosa had been widely resected; subsequent removal of histolocially proven smooth muscle tissue caused the abrupt cessation of receptor discharge. 6. Contraction of airway smooth muscle brought about an increase in receptor discharge frequency. 7. Similar results were found for a smaller number of stretch receptors located in the mainstem and lobar bronchi. 8. These findings procided evidence that stretch receptors are anatomically located within the smooth muscle layer of the airways and are functionally aranged in series with the muscle fibres.     

Model system to study interaction of platelets with damaged arterial wall. II. Inhibition of smooth muscle cell proliferation by dipyridamole and AH-P719

A new in vivo model for the initial events in atherogenesis was employed to investigate drugs which may inhibit intimal muscle cell proliferation following repeated limited endothelial cell injury. An artery forceps was placed over the central artery of the ear of an anesthetized rabbit for 30 min. The forceps were removed, blood flow resumed in the vessel, and platelets contacted the damaged vessel wall. When a vessel was injured two or more times the smooth muscle cells of the media migrated into the intima and proliferated there between 1 and 3 weeks after the last injury despite restoration of an apparently intact endothelium. The intima of control undamaged vessels sometimes contained a few individual smooth muscle cells while vessels injured two, four, or six times showed correspondingly increasing numbers of layers of intimal smooth muscle cells covering increasing amounts of the intima. Arteries from thrombocytopenic rabbits showed, at most, a single layer of smooth muscle cells covering a small area. In rabbits pretreated with dipyridamole (1.5 mg/kg) for 3 days before each injury, proliferation was also limited to a small area. Neither aspirin (8 mg/kg) nor ticlopidine (40 mg/kg, 5X over 3 days), which inhibit platelet aggregation ex vivo, nor the continuous presence of heparin (800 U/kg, bid), reported to inhibit smooth muscle cell growth in vitro and in vivo, prevented smooth muscle cell proliferation in response to two injuries. However, a potent inhibitor of platelet cyclic-adenosine monophosphate (cAMP) phosphodiesterase, AH-P719 (1.5 or 2.1 mg/kg), was able to inhibit intimal smooth muscle cell proliferation in doses that inhibited platelet aggregation ex vivo.     

Ultrastructural localization of peroxidase in atherosclerotic lesions of pigeons.

Atherosclerotic lesions are known to have metabolic alterations which are associated with progressive lipid accumulation. Among the changes, lysosomal enzyme activity has been extensively characterized and at the ultrastructural level has been correlated with the amount of foam cell lipid. In a fashion paralleling lysosomal change, artery wall peroxidase activity is also altered during disease progression. The present study focuses upon the ultrastructural localization of peroxidase activity in atherosclerotic lesions of the aorta and coronary arteries from White Carneau pigeons fed a cholesterol-supplemented (0.3%) diet for 3 years. This resulted in fibrous lesions, rich in smooth muscle cells. The birds were necropsied by perfusion fixation, and peroxidase cytochemistry was carried out using the diaminobenzidine reaction. Peroxidase activity was found within endothelial cells and smooth muscle cells in both the media and intima, but cytochemically demonstrable activity was not found in macrophage foam cells. Peroxidase was localized within the nuclear envelope and endoplasmic reticulum, especially within cells that had lipid inclusions. The degree of peroxidase positivity varied within and among the arteries. In nonlesion regions of the aorta 20% of medial smooth muscle cells was peroxidase positive; the value for coronary artery smooth muscle cells was less. The peroxidase activity within aortic lesions was increased with 44% of intimal smooth muscle cells being positive. Notably, 85-90% of the lipid-containing intimal smooth muscle cells were positive. In contrast, intimal smooth muscle cells in the coronary artery lacked peroxidase reaction product, even in cells containing lipid. We conclude from these studies that aortic lesions contain a cytochemically differentiated subset of lipid-containing, peroxidase-positive smooth muscle cells; but coronary lesions lack a comparable subset of smooth muscle cells.     

Muscle fiber orientation and connective tissue content in the hypertrophied human heart

To elucidate the structural correlates of cardiac failure in myocardial tissue, muscle fiber alignment and connective tissue volume fraction were measured at multiple sites in the left ventricular free wall and in the interventricular septum of 14 human hearts. Group 1 (five hearts; 280 +/- 20 gm.) had no evidence of cardiac disease, group 2 (five hearts; 380 +/- 30 gm.) had a history of systemic hypertension without clinical heart failure, and group 3 (four hearts;; 590 +/- 40 gm.) had both left ventricular overload and congestive failure. Fiber orientations were determined by measuring fiber angle relative to the circumferential direction (helix angle). The fraction of the myocardial volume occupied by connective tissue was determined by point counting. Our results indicate a smooth transition of helix angle from epi- to endocardial surface in the normal left ventricular free wall with nearly 55 per cent of the wall occupied by circumferentially oriented fibers near the cardiac equator (latitude of largest ventricular diameter); morphologically, the interventricular septum was nearly identical with the free wall. Fiber alignment was maintained in all three groups as was the fraction of wall occupied by circumferential fibers. Connective tissue volume fraction was, however, significantly increased (p less than 0.02) in hypertrophied hearts (groups 2 and 3) as compared with normal hearts, and at two of six sites in clinically failed hearts as compared with hypertrophied but functionally compensated hearts. Thus, muscle fiber orientation is not altered in the hypertrophied pressure-overloaded left ventricle, whereas connective tissue content is increased with the increase being greatest in the failing heart.     

The ultrastructure of the various forms of pulmonary arterial intimal fibrosis

Summary Intimal fibrosis of muscular pulmonary arteries may present in various forms and in varying degrees of severity according to the underlying condition. In patients with pulmonary hypertension, the type of intimal fibrosis is often significant with regard to prognosis and reversibility. For these reasons we have studied the ultrastructure of the thickened intimal layer in aged individuals, where intimal fibrosis occurs as a normal age change, and in patients with pulmonary hypertension associated with fibrosis of the lungs, mitral stenosis, chronic pulmonary thromboembolism and plexogenic pulmonary arteriopathy (either primary or secondary to congenital cardiac defects). In all these forms of intimal fibrosis, the cellular component of the subendothelial intimal layer was apparently almost exclusively the smooth muscle cell.These cells usually had a haphazard arrangement. In primary and secondary plexogenic pulmonary arteriopathy, however, there was a more regular circumferential arrangement. The ultrastructural evidence suggested that the intimal cells were derived from medial smooth muscle cells.     

An electron microscopic study of intimal cushions in intracranial arteries of the cat

Subendothelial protuberances associated with the tunica intima have been described by light microscopy at branching sites of arteries in various animal species. These structures are termed intimal cushions or intimal pads. Some investigators regard them as being pathologic, preceding the appearance of atherosclerosis in the vessel wall. This fine structural study was done because of the possible role of intimal cushions in the regulation of cerebral blood flow at branching sites of intracranial arteries. The internal elastic lamina is split at the margins of a cushion to enclose irregularly arranged bundles of smooth muscle cells and collagenous fibrils. These smooth muscle cells are generally arranged at a right angle to smooth muscle cells in the underlying media. The outermost layer of the internal elastic lamina forms a boundary between the body of the cushion and the media; however, areas of discontinuity cause this border to be poorly defined in some instances. Perivascular autonomic nerves do not extend into either media or intimal cushions. Intercellular contacts between smooth muscle cells are observed within cushions as well as in the media, suggesting that cushions may be contractile and capable of modifying cerebral blood flow.     

Time course of the regression of intimal hyperplasia in experimental vein grafts.

A previous study in which vein grafts were removed from the arterial circulation and reimplanted into the venous circulation of the same animal demonstrated regression of vein graft intimal hyperplasia and medial thickening within 14 days. The present study was designed to characterize the kinetics of the morphological and ultrastructural changes over this 14-day period. Twenty-one male New Zealand White rabbits received a reversed vein interposition bypass graft of the right common carotid artery. Fourteen days after the procedure, 21 vein grafts were isolated, removed, and reimplanted into the contralateral external jugular venous system as veno-venous interposition bypass grafts (reversal grafts). The grafts were harvested at 60 minutes, 1 day, 3 days, 5 days, 7 days, and 14 days after reversal. Before insertion into the venous circulation, the vein graft had a confluent endothelial cell surface with multiple layers of smooth muscle cells representing intimal hyperplasia. After 1 hour, the reversal graft retained an intact endothelial cell layer with no evidence of tissue edema or cellular disruption. By 24 hours, there were a few blood cells on the endothelial cell surface. There was no inflammatory infiltrate seen in the subendothelium, and the smooth muscle cells were unaltered. At 3 days, the endothelial cell lining remained intact with no polymorphonucleocytes in the subendothelium or within the graft wall. Underlying smooth muscle cells at this time were noted to contain cytoplasmic vacuoles. At 5 days, there were no inflammatory cells seen on the surface or within the vein graft wall, but many of the underlying smooth muscle cells within the intimal hyperplasia were noted to be fragmented and to have clumping of chromatin. After 7 days, the endothelial cells remained intact and there was widespread evidence of apoptosis beneath the subendothelium with highly fragmented smooth muscle cells, some of which were histologically in the process of breaking up. At 14 days, the grafts retained uniform endothelial cell surfaces. Most of the smooth muscle cells that composed the intimal hyperplasia seen before implantation as a reversal graft were gone. Areas of newly laid down collagen could be observed. There were no acute inflammatory cells but for some mast cells seen in the graft wall. This study demonstrates that in this model, regression of intimal hyperplasia was associated with apoptosis of the smooth muscle cells and the deposition of collagen. There was no evidence that this process is mediated by an acute inflammatory response. Regression therefore appears to be due to induction of smooth muscle cell apoptosis by either a reduction in pressure or flow or a combination of both factors. The findings will enable a systematic cellular and molecular analysis of the biology of regression, which may afford clues to better understand the biology of the developing intimal hyperplasia.     

Rabbit ductus arteriosus during development: Anatomical structure and smooth muscle cell composition

The anatomical structure as well as the smooth muscle cell (SMC¹) composition of the ductus arteriosus (DA) were studied in rabbits ranging in age from 29 days of gestation to 20 days after birth. Computerassisted, three-dimensional reconstructions of hematoxylin-eosin stained serial cryosections from ductus arteriosus-aorta (DA-AO) junctures revealed that DA in animals near term is separated from the aorta by a “septumlike” structure that is continuous with the aortic wall. Two days after birth, obliteration of DA is almost complete, and a small “pocketlike” cavity appears in the pre-existing site in which DA merged into the aorta. This small cavity in the aortic arch was still evident in the large majority of animals examined even 20 days after birth, as also demonstrated by scanning electron microscopy. At this time period DA consisted of a central, fibrotic region surrounded by several layers of SMC (the ligamentum arteriosum, LA) and ended within the aortic media just above the small cavity, forming a round “scar.” Vascular SMC composition of DA during closure was examined by means of indirect and double immunofluorescence procedures, using a panel of monoclonal antibodies against some cytoskeletal and cytocontractile proteins (vimentin, desmin, smooth muscle (SM), and nonmuscle (NM) myosinisoforms). “Intimal cushions” were particularly evident from 5 hr after birth and were found to be desmin-negative, homogenously reactive for vimentin and NM myosin, and heterogeneously stained with anti-SM myosin antibody. In SMC subjacent to the “intimal cushions,” distribution of vimentin and SM myosin was homogeneous, whereas the one of desmin and NM myosin content was heterogeneous. The cytoskeletal and cytocontractile protein content displayed by SMC during the closure of DA is similar to that of “intimal thickening” found in some pathological conditions of the arterial wall in adult rabbits. Completation of DA closure (day 2) was accompanied by the disappearance of cellular heterogeneity in myosin isoform distribution in both the “intimal cushions” and the underlying media. These results give new insights into: (1) the structure of DA-AO juncture, which can be relevant to the physiology of blood circulation in the fetus, and (2) the phenotypic similarity of vascular SMC populations involved in the formation of “intimal cushions” and “intimal thickening”.