The role of vascular smooth-muscle cells in atherogenesis: phenotypic modulation of the medial smooth-muscle cells in the aortic bifurcation.

To elucidate the mechanism of migration of vascular smooth-muscle cells (SMCs) from media to intima, we have investigated the phenotypic modulation of the medial SMC at bifurcation of the celiac artery in 5 children and 3 young persons using a transmission electron microscope. We counted the number of separated SMCs from the elastic layers, although the medial SMCs are fundamentally attached to the elastic fibers, and are still contractile in their phenotypes. Both proximal and distal portions revealed that SMCs in the media were more or less present in the separated state and were ultrastructurally consistent with the synthetic state of SMC in the proximal area and the contractile state in distal areas. In order to migrate from media to intima, medial SMC should separate from the elastic layers and transform their phenotypes. In this paper, we examined the relationship between vascular SMCs and elastic layers in the media and ascertain that it was responsible for the contribution to the subsequent phenotypic modulation and their migration of medial SMCs.     

Distribution of intimal and medial thickening in the human right coronary artery: a study of 17 RCAs

OBJECTIVE: To quantify the distribution of intimal and medial thickening in human right coronary arteries (RCAs) obtained at autopsy. BACKGROUND: The shear and tensile stresses created by arterial bifurcation are believed to result in eccentric fibromuscular intimal thickening that leads to atherosclerosis. Vascular curvature has been cited as a cause of atherosclerosis; however, details of the location and extent of intimal and medial thickness in the largely curved human RCA are not adequately documented. METHODS: The right coronary arteries were obtained from 40 postmortem hearts and cut into 20-30 segments, each being 3-4 mm in length. Microscopic sections from the proximal, acute margin, and distal regions of the RCA were digitized around the circumference of the vessel. Seventeen arteries showed insignificant stenosis (<50%) and were selected for detailed examination. RESULTS: Seventy-one percent (12/17) of proximal sections displayed eccentric intimal thickening. Normalized ensemble averaging revealed a preferential thickening on the myocardial side of the artery. At the acute margin region where curvature is most pronounced and at the distal region, 51% (8/17) of the samples showed eccentric thickening, but the ensemble average thickening in these regions showed no preferential location. In these mildly diseased arteries, the thickened intima comprised of mainly smooth muscle cells with an extracellular matrix of collagen and some elastin. A relatively uniform medial smooth muscle layer was seen at all three locations. CONCLUSIONS: The proximal region of the RCA appears to be a site of intrinsic eccentric intimal thickening with maximum thickness on the myocardial side of the artery. Eccentric thickening does occur in the acute margin and distal regions; however, no distinct pattern or location was evident.     

Immunohistological and ultrastructural analysis of the intimal thickening in coarctation of human aorta.

OBJECTIVES: The histological nature and characteristics of aortic coarctation are not clearly defined, the aim of this study is to analyse intimal thickening in aortic coarctation. METHODS: In order to characterize the components of intimal thickening in coarctation, narrowed segments of aorta obtained after surgery from ten children were examined immunocytochemically and by electron microscopy. RESULTS: Histological analysis of aortic coarctation demonstrated a widened subendothelial region with separation of endothelial cells from the internal elastic lamina. Masson's trichrome staining showed a marked increase in extracellular matrix and cell numbers in the intimal thickening compared with normal aorta. Cellular component analysis demonstrated invagination of the intima by smooth muscle actin-positive cells, with a fragmentation of the internal elastic lamina. No proliferating smooth muscle and inflammatory cells were identified in the intima. In order to characterize the smooth muscle cell phenotypes, various smooth muscle cell markers were sought using specific monoclonal antibodies: alpha-smooth muscle actin, smooth muscle-myosin heavy chain, heavy caldesmon, desmin. In moderate coarcted aorta, at least two distinct smooth muscle phenotypes were identified. In the juxtamedial part of the intima smooth muscle, cells were differentiated and expressed all smooth muscle markers; in the subendothelial part of the intimal thickening, the majority of smooth muscle cells expressed only alpha-smooth muscle actin and appeared dedifferentiated. In regions of marked stenosis, a strong expression of smooth muscle-myosin heavy chain, and heavy caldesmon in the intimal thickening pointed to the presence of redifferentiated smooth muscle cells, not still expressing desmin. Electron microscopic examination also revealed a variety of smooth muscle cell phenotypes in the intimal thickening. In the superficial layer, smooth muscle cells appeared to be in the synthetic state, while in the deeper part, both synthetic and contractile components were identified. CONCLUSIONS: These observations indicated that human coarctation was characterized by intimal recruitment of non-proliferating smooth muscle cells with dedifferentiated phenotype. However, the presence of smooth muscle cells with an intermediate phenotype in the narrowest part of the coarctation suggest that the redifferentiation process could participate in the pathogenesis of aortic coarctation.     

Intimal thickening of jugular and femoral veins vs arteries in the rabbit following investment

The authors induced intimal thickening in the jugular and femoral veins and in the common carotid and femoral arteries of rabbits by placement of a polyethylene tube cuff. The comparative effects on the intima were studied by light and electron microscopy. Even in the veins, thickening resulted from the migration of medial smooth muscle cells into the intima with subsequent proliferation. Thickening in the arteries consisted of tightly packed smooth muscle cells and a few elastic fibers, whereas that in the veins was characterized by an abundance of collagen fibers, layers of smooth muscle cells, and a few elastic fibers. Capillaries were often observed in the thickened intima of the veins but not of the arteries.     

Incidence of myoendothelial gap junctions in the proximal and distal mesenteric arteries of the rat is suggestive of a role in endothelium-derived hyperpolarizing factor-mediated responses

Although the chemical nature of endothelium-derived hyperpolarizing factor (EDHF) remains elusive, electrophysiological evidence exists for electrical communication between smooth muscle cells and endothelial cells suggesting that electrotonic propagation of hyperpolarization may explain the failure to identify a single chemical factor as EDHF. Anatomical evidence for myoendothelial gap junctions, or the sites of electrical coupling, is, however, rare. In the present study, serial-section electron microscopy and reconstruction techniques have been used to examine the incidence of myoendothelial gap junctions in the proximal and distal mesenteric arteries of the rat where EDHF responses have been reported to vary. Myoendothelial gap junctions were found to be very small in the mesenteric arteries, the majority being <100 nm in diameter. In addition, they were significantly more common in the distal compared with the proximal regions of this arterial bed. Pentalaminar gap junctions between adjacent endothelial cells were much larger and were common in both proximal and distal mesenteric arteries. These latter junctions were frequently found near the myoendothelial gap junctions. These results provide the first evidence for the presence of sites for electrical communication between endothelial cells and smooth muscle cells in the mesenteric vascular bed. Furthermore, the relative incidence of these sites suggests that there may be a relationship between the activity of EDHF and the presence of myoendothelial gap junctions.     

Role of endothelial cells in relaxation of isolated arteries by bradykinin.

Bradykinin elicits relaxation of isolated transverse rings of canine coronary, celiac, superior mesenteric, renal, splenic, pulmonary, gastric, and femoral arteries. After endothelial cells of the vessel wall are removed by rubbing of the intimal surface, canine arteries fail to relax upon addition of bradykinin. The endothelium-dependent relaxation of canine arteries remains intact after treatment with cyclooxygenase inhibitors (indomethacin and flurbiprofen), and this argues against mediation by prostaglandins. When they are stimulated with bradykinin, endothelial cells of canine arteries appear to release a substance mediating vascular smooth muscle relaxation. In contrast, preparations of arteries of cats (superior mesenteric) and rabbits (superior mesenteric and celiac) may be rubbed on the intimal surface without a consistent loss of sensitivity to the relaxing effects of bradykinin. In addition, relaxation of the cat and rabbit arteries is completely blocked by cyclooxygenase inhibitors. Preliminary studies indicate that bradykinin relaxes human arteries in an endothelium-dependent manner and that this effect is not mediated by prostaglandins. We have previously reported that arteries of all species tested require the presence of endothelial cells for relaxation in response to acetylcholine and we have also demonstrated, using the rabbit aorta, that this effect is mediated by the release of an uncharacterized substance from these cells that relaxes vascular smooth muscle. We conclude that bradykinin relaxes canine and human arteries via a similar mechanism but that it relaxes cat and rabbit arteries by stimulating release of prostaglandins from as yet undefined cell types.     

Atherosclerosis in the hypercholesterolemic hare. Comparison of coronary artery lesions induced by dietary cholesterol in the hare and the rabbit

Atherosclerotic lesions in coronary arteries were compared in 10 hybrid hares and 14 rabbits after induction of hypercholesterolemia, using a cholesterol-enriched diet. All proximal portions of hare coronary arteries contained intimal lesions, often with severe luminal stenosis. These lesions were characterized by the presence of foam cells, smooth muscle cells, and areas of atheronecrosis. Foam cells were also found focally in the media. As part of the intimal changes, iron deposition was present in 65% and calcification was present in 32.5% of proximal segments examined. The proportion of segments with intimal lesions and the intima/media cross-sectional area ratios (I/M ratios) were greatest in proximal segments with stepwise decreases in the mid and distal segments. As area of myocardial infarction was present in one hare. In contrast, 46.5% of proximal segments of rabbit coronary arteries had no intimal lesions and those lesions present had no calcium or iron deposition. No infarction was observed in rabbit hearts. The proportion of segments with lesions and the mean I/M ratios were significantly greater in the hare than the rabbit, with proximal and mid coronary segments showing the most marked differences. The hare appears to develop coronary artery lesions more like those seen in man, with high grade, proximal stenoses occurring uniformly in hypercholesterolemic animals. In contrast, the atherosclerosis developing in rabbit coronary arteries is less uniform and includes involvement of intramyocardial arterioles. The hare offers several advantages as a model of human atherosclerosis.     

Distribution of Type I,III, IV and V Collagen in Normal and Atherosclerotic Human Arterial Wall: Immunomorphological Characteristics

35 autopsies — aged 30 to 75 years — were investigated in order to establish trends of collagen localization in various types of arteries depending on age, arterial size and degree of atherosclerosis. Cryostat sections stained with highly specific antibodies to human types I, III, IV or V collagen, or with the antiserum to smooth muscle myosin were examined by the indirect immunofluorescence technique.Localization of type III collagen was very similar to that of type I. Fibrous structures of both type I and type III were then major constituents of the intima, media and adventitia. Sparse fibrils of type I and type III collagens were revealed in the subendothelium of unaffected intima. They gradually became abundant in the deeper intimal layers contrasting with loose fibrillar formations of the media. The content of interstitial collagens was significantly increased in the subendothelium of local intimal thickenings and in a thickened intima of the aged. This fact, considering the thrombogenicity of interstitial collagens, may be relevant to the atherogenesis through the "response-to-injury" mechanism. Type IV and type V collagens are localized to the endothelial basement membrane and basement membranes of smooth muscle cells of the intima and media. Diffusely distributed type V collagen was also observed in the intercellular space of the intimaIn lipid streaks, parallel layers of condensed interstitial collagens separated groups of cells and extracellular lipid depositions. In fibrous plaques, types I and III became prevalent structural elements and their densely packed fibers occupied whole regions devoid of any type IV and type V collagen. Heavily thickened type IV collagen structures surrounding individual smooth muscle cells were found in fibrous plaques, but never, in unaffected intima.     

Differential expression of connexin43 and desmin defines two subpopulations of medial smooth muscle cells in the human internal mammary artery.

Upregulation of connexin43-gap junctions is associated with transition of contractile vascular smooth muscle cells (SMCs) to the synthetic state. To determine whether phenotypically distinct subpopulations of medial SMCs differentially express connexin43, we investigated the human distal internal mammary artery, a structurally heterogeneous vessel with features ranging from elastic to elastomuscular to muscular. Immunoconfocal microscopy combined with quantitative analysis and complemented by in situ hybridization showed that SMCs in the elastic medial regions expressed high levels of connexin43 but low levels of desmin, whereas those of muscular medial regions expressed low levels of connexin43 but high levels of desmin. Ultrastructurally, SMCs of both regions were of the contractile phenotype, but the former cells were irregular in shape with relatively prominent synthetic organelles whereas the latter were spindle shaped with fewer synthetic organelles. Vimentin, smooth muscle alpha-actin, calponin, h-caldesmon, and myosin heavy chains (SM1 and SM2) were equally highly expressed by most cells in both subpopulations. The connexin43/desmin expression pattern of SMCs in regions of intimal thickening resembled those of elastic medial regions. These findings refine the view suggested from previous studies that high levels of connexin43 expression are associated with SMCs of a less contractile/more synthetic phenotype. In the internal mammary artery, the 2 subpopulations of SMCs with markedly different connexin43 expression levels both represent a differentiated contractile phenotype, but the subpopulation showing high levels of connexin43-gap junctions is characterized by low levels of desmin and structural features that reflect a more synthetic tendency.     

The calcium antagonist nifedipine inhibits arterial smooth muscle cell proliferation

Migration of smooth muscle cells from the media to the intima of the arterial wall and proliferation of intimal smooth muscle are major early events in the formation of an atherosclerotic lesion. The start of proliferation requires that the cells have passed through a modulation from contractile to synthetic phenotype and that they are stimulated with growth factors. Here, we have examined the effects of the calcium antagonist nifedipine on phenotypic modulation and growth of isolated rat arterial smooth muscle cells cultivated in vitro. The results indicate that micromolar concentrations of nifedipine slow down the rate of transformation of the cells from a contractile to a synthetic phenotype and inhibit initiation of DNA synthesis as well as cellular proliferation. The inhibitory effect on DNA synthesis was seen both in cells stimulated with whole blood serum and with purified platelet-derived growth factor. The results raise the possibility that nifedipine may be used to prevent atherogenesis and to inhibit progression of fibromuscular lesions by interfering with the proliferation of arterial smooth muscle cells.     

Macrophages, macrophage foam cells, and eccentric intimal thickening in the coronary arteries of young children

We surveyed the incidence and location of macrophages and macrophage foam cells in the coronary artery intima of 63 children that died in the first 5 years of life. We related the data on macrophages and macrophage foam cells to intimal smooth muscle cells and to measurements of intima:media area and thickness. All morphometric data were obtained from coronary arteries that were fixed by perfusion with glutaraldehyde under pressure, embedded in Maraglas, and cut into 1-micron cross-sections, and 65-nm fine sections. Coronary artery intima was always thicker (eccentric thickening) at bifurcations in the half of the circumference opposite to the flow divider. This was true for both male and female children. The remaining part of the coronary artery intima was less thick (diffuse thickening). Both types of intimal thickening were composed of an inner layer in which glycosaminoglycan ground substance predominated and a deeper musculoelastic layer. Fifty-nine children (94%) had intimal macrophages. Twenty children also had macrophage foam cells. Of 33 children aged to 8 months, 15 (45%) had macrophage foam cells. Of the 30 children older than 8 months, 5 (17%) had macrophage foam cells. Macrophages and macrophage foam cells occurred in the GAG-rich layer of the intima as isolated cells. In 5 infants macrophage foam cells occurred also as clusters of many cells. Macrophages were more numerous in cases that also had macrophage foam cells. Macrophages were 6 times, and macrophage foam cells 5 times more numerous in eccentric intimal thickening than in diffuse intimal thickening.     

Transplant Vasculopathy

Transplant vasculopathy constitutes the major impediment to long-term survival in heart transplant recipients. Within the “response to immune injury” paradigm, it can best be understood as the resultant of an orchestrated recipient immune response to the initial allogenic stimulus by graft vascular endothelium. This response incorporates the elaboration of complex coordinated cytokine patterns and corresponding cell types including B-lymphocytes, T^_helperl_ and T^_helper2_cells, cytotoxic T-cells, macrophages, and polymorphonuclear cells. These attack the alloantigenic vascular endothelium and lead, by complex cytokine signalling, to migration of donor smooth muscle cells from the media into the intima, associated with a switch from the contractile to a synthetic phenotype. In conjunction with recipient T-cells, macrophages, and lipids, the intimal fibroproliferative growth of the donor vessel is hereby initiated.     

Vascular changes at the prehypertensive phase in the mesenteric arteries from spontaneously hypertensive rats

Morphometric measurements of three categories of mesenteric vessels (representing elastic, muscular and arteriolar vessels) from prehypertensive spontaneously hypertensive rats (SHR) and age-matched Wistar-Kyoto rats (WKY) were carried out at the light and electron microscope levels. Structural alterations of the blood vessels were already present in the SHR, even though the blood pressure was not yet elevated as compared with age-matched WKY. No change was found in the elastic vessels (superior mesenteric artery). Among the muscular arteries (i.e. large mesenteric arteries), the increase in vessel wall cross-sectional area was due to the increase in the intima, media and adventitia. Increase in media was due to hyperplasia of the smooth muscle cells. The smooth muscle cells were not hypertrophied. Nerve density was also higher in the large mesenteric arteries of SHR. In the arteriolar vessels (i.e. small mesenteric arteries), wall to lumen ratio, as well as media to lumen ratio, were increased in the SHR. The number of smooth muscle cell layers was also increased. In all these vessel types, the cross-sectional area of the lumen under maximal relaxation was similar between SHR and WKY, except in small mesenteric arteries where the lumen was smaller in the SHR. Our results suggest that structural alteration of the blood vessels at the prehypertensive phase may be one of the contributing factors leading to the development of hypertension in the SHR.     

Changes of coronary artery morphology and distribution of smooth muscle cells during progression of coronary atherosclerosis after heart transplantation

To examine the changes in coronary artery morphology and the distribution of smooth muscle cells during the progression of coronary atherosclerosis after cardiac transplantation, intimal and medial tissues were evaluated and the density of smooth muscle cells in the media were measured 30 and 60 days after transplantation by microspectrophotometry from rats receiving both auto- and allo-transplantation. Transplanted animals were given cyclosporine A to prevent graft rejection. Signs of rejection were not seen in the animals receiving auto-transplants. Rejection gradually progressed after transplantation in animals receiving allografts. The intima of the coronary arteries in the allograft group was significantly thickened at both 30 and 60 days after transplantation. The total intimal area in the day 60 group was significantly increased relative to the day 30 group among animals receiving allo-transplantation. The medial area of the coronary arteries in the group receiving allotransplantation was significantly less than that of the auto-transplantation group at both 30 and 60 days after transplantation. Azocarmine G stain revealed that the principal component of the thickened intima was smooth muscle cells. Coronary arteries in the allotransplantation group had disruption of the internal elastic lamina. We therefore hypothesized that the smooth muscle cells (SMCs) in the intima are probably derived from the media. The density of SMCs in the media was measured by microspectrophotometry. The density of SMCs was significantly decreased in the allo-transplantation group relative to the autotransplantation group. We conclude that intimal thickening of the coronary arteries is due to SMC proliferation, the myocytes being from the media through disruption of the internal elastic lamina. This process is similar to the mechanism of the development of atherosclerosis.     

Immunocytochemical analysis of the atherosclerotic lesion.

We have performed immunocytochemical investigations on the distribution of various cell types and proliferating cells in human atherosclerotic lesions. Studies include fibrocellular tissue response following percutaneous transluminal coronary angioplasty (PTCA) or aortocoronary (A-C) bypass operation using monoclonal antibodies specific to smooth muscle cells, macrophages, endothelial cells, lymphocytes and proliferating cell nuclear antigen (PCNA). All studies were performed on methanol-Carnoy's-fixed, paraffin-embedded tissues. The cellular composition of the following three types of raised lesions were analyzed: 1) fibro-fatty lesions composed almost exclusively of macrophages; 2) fibrous lesions predominantly composed of smooth muscle cells; 3) advanced plaques characterized by complex layers of smooth muscle cells and macrophages with considerable variation from region to region. Also noted were foci of medial and even intimal vascularization subjacent to the more advanced plaques. Cells encountered within the fibrous intimal thickening in the vein graft or fibrocellular tissue response following PTCA were predominantly smooth muscle cells in origin. Some cells were PCNA-positive. These studies demonstrate the application of monoclonal antibody technology to the study of the cellular composition and cell kinetics of human atherosclerotic lesions.     

Accumulated endogenous nitric oxide synthase inhibitors, enhanced arginase activity, attenuated dimethylarginine dimethylaminohydrolase activity and intimal hyperplasia in premenopausal human uterine arteries

The present study was designed to investigate the involvement of nitric oxide synthase (NOS), endogenous NOS inhibitors, arginase, which shares L-arginine as a common substrate with NOS, and dimethylarginine dimethylaminohydrolase (DDAH) as a metabolizing enzyme of NOS inhibitors in the occurrence of intimal hyperplasia in premenopausal human uterine arteries. Fifty-two uterine arteries were obtained from 52 patients undergoing total hysterectomy with an informed consent for the present study. All specimens were assessed histologically and the intima:media ratio (%) was evaluated as an index of intimal hyperplasia. Nineteen specimens were found to be histologically normal (intima:media ratio=16.1+/-0.8%), whereas remaining 33 specimens were categorized as intimal hyperplasia (intima:media ratio=34.4+/-1.5%). The intimal hyperplasia was associated with the impaired cyclic GMP production without change in endothelial NOS activity per se, accumulation of endogenous NOS inhibitors in endothelial cells, attenuated DDAH activity in endothelial cells and enhanced arginase activity in endothelial cells and smooth muscle layer. These findings suggest that the impaired cyclic GMP production as a marker of NO production is possibly due to the accumulated endogenous NOS inhibitors and enhanced arginase activity, which, in turn, closely relates to the occurrence of intimal hyperplasia, and that the impaired DDAH activity would result in the accumulation of endogenous NOS inhibitors in endothelial cells. Because of the enhanced arginase activity in endothelial cells and smooth muscle layer, the accelerated polyamine biosynthetic pathway may be implicated in the occurrence of intimal hyperplasia in premenopausal human uterine arteries.     

Neonatal intima formation in the human coronary artery.

Intimal masses develop in the human coronary arteries of all humans, becoming atherosclerotic in later life either because of focal accumulation of lipid or the resulting response to injury. We evaluated the time course of formation of the intimal mass in the proximal left anterior descending coronary artery in autopsy specimens from 91 patients between 17 weeks' gestation and 23 months of postnatal age. Intima was rarely found before 30 weeks' gestation; however, the frequency with which at least some intimal cells were observed increased to 35% between 36 weeks' gestation and birth. By 3 months after birth, all patients had an intimal mass at this coronary location. The mean intima/media ratio was 0.1 just after birth and increased continuously to the second postnatal year. Replication of medial smooth muscle cells, indicated by proliferating cell nuclear antigen staining, was high before birth and decreased between birth and 2 years of age. However, the replication index of the intima remained at 2% to 5%. Thus, coronary intimal cells appearing in the perinatal period may arise by migration after replication of medial smooth muscle, as is seen in models of carotid artery balloon injury. In conclusion, formation of the coronary artery intima is a rapid process, beginning in the peripartum or postpartum period. Given the clonality of the adult lesion and the lack of proliferation in later stages of lesion formation, it is intriguing to speculate that this event may form the basis for atherosclerosis in later life.     

The distribution and production of cholesteryl ester transfer protein in the human aortic wall

Cholesteryl ester transfer protein (CETP) has been considered to mediate the transfer of cholesteryl ester from arterial wall, however, the distribution and production of CETP in human arterial wall remains unclear. Present study histopathologically demonstrated the distribution of CETP and CETP mRNA in the human aortic wall by immunohistochemistry and in situ hybridization. While CETP was constantly distributed in the media, the protein was recognized within the intima with fibrocellular thickening and atherosclerotic intima. Double immunostaining methods demonstrated CETP expression in smooth muscle cells in the intima and media. CETP mRNA was detected not only in intimal cells but medial smooth muscle cells. Intimal cells expressing CETP mRNA were considered to be monocyte-derived macrophages and smooth muscle cells by immunohistochemistries using two antibodies against smooth muscle actin and human macrophage on the subserial sections. Our in vivo study provides that CETP is produced by smooth muscle cells in the intima and media of human aorta, and it is suggested that arterial smooth muscle cells positively participate in the removal of excessive cholesteryl ester from the arterial wall by CETP production.     

Measurements from light and polarised light microscopy of human coronary arteries fixed at distending pressure

With the long term goal of improving our understanding of the mechanisms involved in coronary artery spasm, we have undertaken a two part study of the artery structure. We have made a comparison of the relative proportions of the different layers in proximal and distal regions of the main coronary arteries, and have quantitatively assessed their three dimensional structural fabric. Major coronary arteries from nine hearts were prepared for histological examination after fixation at a transmural pressure of 120 mm Hg. Measurements from 14 proximal and distal pairs of the cross sectioned arteries showed a dominant subendothelial layer, which diminished in thickness distally, with a small fraction of muscle cells interspersed with collagen. Three dimensional orientation measurements of the collagen and muscle components, which are birefringent, were obtained from one pair of segments from each of the left anterior descending, circumflex and right coronary arteries, using the polarising light microscope and Universal stage. Findings showed (1) a single circumferential order of adventitial collagen, with a mean circular standard deviation (CSD) of 22.3 degrees; (2) very highly ordered medial smooth muscle, mean CSD of 5.0 degrees (both findings are quantitatively similar between proximal and distal segments of artery, and between arteries); and (3) a multilayered fabric of collagen in the subendothelium in all vessel segments. The principal contributor to functional differences between proximal and distal regions may be the prominent and structurally varied subendothelial layer of the coronary arteries.