Presence Of Smooth Muscle Cells Research Articles

Ligamentum Arteriosum: An Innervated and Contractile Muscular Structure

IntroductionThe ligamentum arteriosum (LA) is generally described as a mere fibrotic remnant of the embryonic bypass (ductus arteriosus) from the pulmonary trunk to the aortic arch, obliterating soon after childbirth. There have been anecdotal and contradictory reports on this speculated morphological change. This study set out to elucidate the morphology, innervation, neurochemistry and function of LA.MethodLA of human, pig, wild‐type and appropriate transgenic reporter mouse strains were studied using routine and special histological staining methods, single‐ and double‐immunofluorescence labeling using antibodies directed against structural markers such as α‐smooth muscle actin (αSMA) and protein gene product 9.5 (PGP 9.5), against neuropeptides such as neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), substance P (SP), and calcitonin gene‐related peptide (CGRP), and against transmitter synthesizing enzymes such as tyrosine hydroxylase (TH; noradrenergic), choline acetyltransferase (ChAT; cholinergic), and nitric oxide synthase (NOS). Additionally, pig and mice LA were investigated using transmission electron microscopy (TEM). Organ bath experiments subjected pig LA to increasing cumulative doses of exogenous noradrenalin (NA), ɑ1‐adrenergic antagonists (tamsulosin and prazosin) and varying Hz of electrical field stimulation.ResultsContrary to a canonical ligament, the LA was mainly made up by αSMA‐positive cells in all three species. TEM confirmed the presence of smooth muscle cells with caveolae, mmyofilaments’, dense bands etc. within the LA. The ligament received a noticeable amount of noradrenergic (TH, NPY) and sensory (CGRP, SP) but no cholinergic innervation in all three species investigated. The LA exhibited a dose‐ and frequency‐dependent contraction in response to cumulative doses of exogenous NA and electrical field stimulation respectively. Furthermore, NA pre‐contracted LA relaxed to baseline upon administration of ɑ1‐adrenergic antagoniststs (tamsulosin and prazosin).ConclusionThe LA may not function in its original capacity as a foetal shunt but still retains smooth muscles cells until senescence. Ultra‐structurally, the presence of myofilaments, dense bodies and dense bands on smooth muscle cells (SMC) observed within the LA are findings compatible with the apparatus responsible for contraction in SMC. Furthermore, the presence of numerous mitochondria, a cellular organelle, and a potential site of calcium accumulation for smooth muscle contraction, observed within the LA may play a role in its contractility. Immunoreactivity to TH‐, NPY‐, SP‐, and CGRP‐ is suggestive of sympathetic and sensory innervation within the LA. Additionally, dose‐response contractility and relaxation of pre‐contracted LA to exogenous NA and ɑ1‐adrenergic antagonists in organ bath experiments suggests activity of ɑ1‐adrenoceptors. Conclusively, it is evident that LA is made up of contractile SMC with noradrenergic and sensory innervation. Its contractility may influence the compliance or impedance of the two major vascular segments to which the LA is attached.

Smooth muscle cells affect differential nanoparticle accumulation in disturbed blood flow-induced murine atherosclerosis.

Atherosclerosis is a lipid-driven chronic inflammatory disease that leads to the formation of plaques in the inner lining of arteries. Plaques form over a range of phenotypes, the most severe of which is vulnerable to rupture and causes most of the clinically significant events. In this study, we evaluated the efficacy of nanoparticles (NPs) to differentiate between two plaque phenotypes based on accumulation kinetics in a mouse model of atherosclerosis. This model uses a perivascular cuff to induce two regions of disturbed wall shear stress (WSS) on the inner lining of the instrumented artery, low (upstream) and multidirectional (downstream), which, in turn, cause the development of an unstable and stable plaque phenotype, respectively. To evaluate the influence of each WSS condition, in addition to the final plaque phenotype, in determining NP uptake, mice were injected with NPs at intermediate and fully developed stages of plaque growth. The kinetics of artery wall uptake were assessed in vivo using dynamic contrast-enhanced magnetic resonance imaging. At the intermediate stage, there was no difference in NP uptake between the two WSS conditions, although both were different from the control arteries. At the fully-developed stage, however, NP uptake was reduced in plaques induced by low WSS, but not multidirectional WSS. Histological evaluation of plaques induced by low WSS revealed a significant inverse correlation between the presence of smooth muscle cells and NP accumulation, particularly at the plaque-lumen interface, which did not exist with other constituents (lipid and collagen) and was not present in plaques induced by multidirectional WSS. These findings demonstrate that NP accumulation can be used to differentiate between unstable and stable murine atherosclerosis, but accumulation kinetics are not directly influenced by the WSS condition. This tool could be used as a diagnostic to evaluate the efficacy of experimental therapeutics for atherosclerosis.

High lipoprotein(a) is associated with major adverse limb events after femoral artery endarterectomy

Backgrounds and aimsElevated lipoprotein(a) (Lp[a]) has been identified as a causal risk factor for cardiovascular disease including peripheral arterial disease (PAD). Although Lp(a) is associated with the diagnosis of PAD, it remains elusive whether there is an association of Lp(a) with cardiovascular and limb events in patients with severe PAD. MethodsPreoperative plasma Lp(a) levels were measured in 384 consecutive patients that underwent iliofemoral endarterectomy and were included in the Athero-Express biobank. Our primary objective was to assess the association of Lp(a) levels with Major Adverse Limb Events (MALE). Our secondary objective was to relate Lp(a) levels to Major Adverse Cardiovascular Events (MACE) and femoral plaque composition that was acquired from baseline surgery. ResultsDuring a median follow-up time of 5.6 years, a total of 225 MALE were recorded in 132 patients. Multivariable analysis, including history of peripheral intervention, age, diabetes mellitus, end stage renal disease and PAD disease stages, showed that Lp(a) was independently associated with first (HR of 1.36 (95% CI 1.02–1.82) p = .036) and recurrent MALE (HR 1.36 (95% CI 1.10–1.67) p = .004). A total of 99 MACE were recorded but Lp(a) levels were not associated with MACE.sLp(a) levels were significantly associated with a higher presence of smooth muscle cells in the femoral plaque, although this was not associated with MALE or MACE. ConclusionsPlasma Lp(a) is independently associated with first and consecutive MALE after iliofemoral endarterectomy. Hence, in patients who undergo iliofemoral endarterectomy, Lp(a) could be considered as a biomarker to enhance risk stratification for future MALE.

In vivo application of decellularized rat colon and evaluation of the engineered scaffolds following 9 months of follow-up.

The aim of this study was to investigate the rat small intestine mesentery and colon as natural bio-reactors for rat colon-derived scaffolds. We decellularized eight whole rat colons by a perfusion-based protocol using 0.1% sodium dodecyl sulfate for 24 hr. The provided bio-scaffolds were examined by histological staining, scanning electron microscopy, and collagen and sulfated glycosaminoglycan quantification. Subsequently, we implanted 4 cm segments of the provided bio-scaffolds into two groups of animal models comprising tissue grafting into the mesenteric tissue (n: 10) and end-to-end anastomosis (n: 10) to the colon of host rats. Following 9 months of follow-up, we harvested the grafts and performed histological and immunohistochemical studies as well as real-time PCR evaluation for telomerase activity of the samples. Histological staining, scanning electron microscopy and protein content evaluation of the acellular tissues confirmed the complete removal of the cellular components and preservation of the extracellular matrix. Histopathological assessment of the implanted scaffolds was suggestive of a regenerative process in both groups. Moreover, immunohistochemical analysis of the samples confirmed the presence of smooth muscle cells, endothelial progenitor cells, and neural elements in both groups of grafted scaffolds. Our data confirmed the recellularization of the acellular colon grafts in both groups after 9 months of follow up. Also, the implanted tissues demonstrated different characteristics based on their implantation location. The outcomes of this investigation illustrate the capability of acellular tissues for in vivo application and regeneration.

Proximal ureteral reconstruction using renal capsule flap: a canine experimental model.

IntroductionThe aim of this article was to evaluate the effectiveness of using the renal capsule in ureteral reconstruction in a canine model.Material and methodsTen clinically healthy male adult dogs were used in this study. Dogs underwent ureteral reconstruction using a tube-shaped flap of the renal capsule.ResultsAll but one animal (90%) survived till nephrectomy and thereafter. At 30 days after operation, the double-J stent was removed from the ureter, and at the 60th day, intravenous pyelography confirmed openness of the duct. The internal surface of the tunneled flap was coated with thick, folded urothelium. Maturing granulation tissue and angiogenesis as well as fiber producing fibroblasts were observed in the lamina propria. The presence of smooth muscle cells beneath the lamina propria indicated complete reconstitution of the damaged ureter.ConclusionsThe results showed that the autologous renal capsular flap provided a practical option for treating ureteral defects in dogs with an acceptable outcome. So, using the selfsame renal capsular tissue is a feasible method for restoration of the injured proximal ureter.

Toward the Optimization of Dinitrosyl Iron Complexes as Therapeutics for Smooth Muscle Cells.

In this study, dinitrosyl iron complexes (DNICs) are shown to deliver nitric oxide (NO) into the cytosol of vascular smooth muscle cells (SMCs), which play a major role in vascular relaxation and contraction. Malfunction of SMCs can lead to hypertension, asthma, and erectile dysfunction, among other disorders. For comparison of the five DNIC derivatives, the following protocols were examined: (a) the Griess assay to detect nitrite (derived from NO conversion) in the absence and presence of SMCs; (b) the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium (MTS) assay for cell viability; (c) an immunotoxicity assay to establish if DNICs stimulate immune response; and (d) a fluorometric assay to detect intracellular NO from treatment with DNICs. Dimeric Roussin's red ester (RRE)-type {Fe(NO)2}9 complexes containing phenylthiolate bridges, [(μ-SPh)Fe(NO)2]2 or SPhRRE, were found to deliver NO with the lowest effect on cell toxicity (i.e., highest IC50). In contrast, the RRE-DNIC with the biocompatible thioglucose moiety, [(μ-SGlu)Fe(NO)2]2 (SGlu = 1-thio-β-d-glucose tetraacetate) or SGluRRE, delivered a higher concentration of NO to the cytosol of SMCs with a 10-fold decrease in IC50. Additionally, monomeric DNICs stabilized by a bulky N-heterocyclic carbene (NHC), namely, 1,3-bis(2,4,6-trimethylphenyl)imidazolidene (IMes), were synthesized and yielded the DNIC complexes SGluNHC, [IMes(SGlu)Fe(NO)2], and SPhNHC, [IMes(SPh)Fe(NO)2]. These oxidized {Fe(NO)2}9 NHC DNICs have an IC50 of ∼7 μM; however, the NHC-based complexes did not transfer NO into the SMC. Per contra, the reduced, mononuclear {Fe(NO)2}10 neocuproine-based DNIC, neoDNIC, depressed the viability of the SMCs, as well as generated an increase of intracellular NO. Regardless of the coordination environment or oxidation state, all DNICs showed a dinitrosyl iron unit (DNIU)-dependent increase in viability. This study demonstrates a structure-function relationship between the DNIU coordination environment and the efficacy of the DNIC treatments.

Lipid core nanoparticles as vehicle for docetaxel reduces atherosclerotic lesion, inflammation, cell death and proliferation in an atherosclerosis rabbit model

Chemotherapeutic agents used in cancer treatment associated to nanoparticles (LDE) that mimic the composition of low-density lipoprotein and buffer their toxicity can have strong anti-atherosclerosis action, as we showed in cholesterol-fed rabbits. Here, a novel preparation of docetaxel (DTX) carried in LDE was evaluated. Eighteen rabbits were fed 1% cholesterol during 8 weeks. After the first 4 weeks, 9 animals were treated for 4 weeks with intravenous LDE-DTX (1 mg/kg/week) and 9 with LDE only (controls) once a week for 4 weeks. Animals were then euthanized and the aortas were analyzed for morphometry, immunohistochemistry and Western blot. LDE-DTX treated group showed 80% reduction of atheroma area compared to controls. LDE-DTX treatment reduced in 60% the protein expression of macrophage marker CD68 and of MCP-1 in 80%. LDE-DTX pronouncedly lowered expression of pro-inflammatory markers NF-κB, TNF-α, IL-1β, IL-6 and von Willebrand factor and elicited 40% reduction in cell proliferation marker PCNA. The presence of smooth muscle cells in the intima was 85% smaller than in controls. Pro-apoptotic caspase 3, caspase 9, Bax, and anti-apoptotic Bcl-2 all were reduced by LDE-DTX. Protein expression of MMP-2 and MMP-9, TGF-β, and collagen 1 and 3 were also markedly lowered by the LDE-DTX treatment. Animals showed no hematological, hepatic or renal toxicity consequent to LDE-DTX treatment. In conclusion, LDE-DTX showed a wide array of strong effects on pro-inflammatory and proliferation-promoting factors that drive the lesion development. These findings and the lack of observable toxicity indicate that LDE-DTX can be a candidate for future clinical trials.

Anatomical and Histological Study of the Human Neonate Lungs

The current study aims to describe the normal anatomical structures of neonate lungs )weights, volumes and dimensions( as well as the architectural histology of structure In this study, 20 neonate cadavers collected with age ranging from 0-28 days, all these cadavers were collected from the Forensic Medicine in Baghdad and Kirkuk. Following dissection, the organ weight and gross metrical measurements were measured. Histological study was done on all cadavers enrolled in the study using Periodic Acid Schiff )PAS( Stain The results showed that the weight of neonatal lung was increased proportionally with age and the highest mean weight, volume and dimensions of lungs were found in neonates whose in the age group 22-28 day. The study revealed that dimensions of the left lungs were slightly more than those of right lungs especially in the age group 22-28 days. The study indicated that the volume of neonatal lungs were increased proportionally with age and the highest mean volume of lungs was found in neonates whose in the age group 22-28 days 50.03 cm3. The tunica media of the blood vessels of small and medium sized were well demonstrated of the thickness of alveolar walls also were obvious with inflammatory cell infiltration, the interstitial C.T. also associated with presence of smooth muscle cells, surrounded by segments of hyaline cartilage which are demonstrated easily, the lumen of bronchus appeared containing exudate, around the lung was containing with collagen bundle the elastic fibers. The basement membrane of the pulmonary blood vessels was demonstrated well by PAS which appeared thickened and the lumen of blood vessels was filled with blood serum and individual inflammatory cell. The interstitial C.T. around the blood vessels were containing dust cell with lymphocyte infiltration. .

Contractile Smooth Muscle and Active Stress Generation in Porcine Common Carotids.

The mechanical response of intact blood vessels to applied loads can be delineated into passive and active components using an isometric decomposition approach. Whereas the passive response is due predominantly to the extracellular matrix (ECM) proteins and amorphous ground substance, the active response depends on the presence of smooth muscle cells (SMCs) and the contractile machinery activated within those cells. To better understand determinants of active stress generation within the vascular wall, we subjected porcine common carotid arteries (CCAs) to biaxial inflation-extension testing under maximally contracted or passive SMC conditions and semiquantitatively measured two known markers of the contractile SMC phenotype: smoothelin and smooth muscle-myosin heavy chain (SM-MHC). Using isometric decomposition and established constitutive models, an intuitive but novel correlation between the magnitude of active stress generation and the relative abundance of smoothelin and SM-MHC emerged. Our results reiterate the importance of stretch-dependent active stress generation to the total mechanical response. Overall these findings can be used to decouple the mechanical contribution of SMCs from the ECM and is therefore a powerful tool in the analysis of disease states and potential therapies where both constituent are altered.

Detailed muscular structure and neural control anatomy of the levator ani muscle: a study based on female human fetuses

Injury to the levator ani muscle or pelvic nerves during pregnancy and vaginal delivery is responsible for pelvic floor dysfunction. We sought to demonstrate the presence of smooth muscular cell areas within the levator ani muscle and describe their localization and innervation. Five female human fetuses were studied after approval from the French Biomedicine Agency. Specimens were serially sectioned and stained by Masson trichrome and immunostained for striated and smooth muscle, as well as for somatic, adrenergic, cholinergic, and nitriergic nerve fibers. Slides were digitized for 3-dimensional reconstruction. One fetus was reserved for electron microscopy. We explored the structure and innervation of the levator ani muscle. Smooth muscular cell beams were connected externally to the anococcygeal raphe and the levator ani muscle and with the longitudinal anal muscle sphincter. The caudalmost part of the pubovaginal muscle was found to bulge between the rectum and the vagina. This bulging was a smooth muscular interface between the levator ani muscle and the longitudinal anal muscle sphincter. The medial (visceral) part of the levator ani muscle contained smooth muscle cells, in relation to the autonomic nerve fibers of the inferior hypogastric plexus. The lateral (parietal) part of the levator ani muscle contained striated muscle cells only and was innervated by the somatic nerve fibers of levator ani and pudendal nerves. The presence of smooth muscle cells within the medial part of the levator ani muscle was confirmed under electron microscopy in 1 fetus. We characterized the muscular structure and neural control of the levator ani muscle. The muscle consists of a medial part containing smooth muscle cells under autonomic nerve influence and a lateral part containing striated muscle cells under somatic nerve control. These findings could result in new postpartum rehabilitation techniques.

Development of the Ex Vivo Mouse Mesometrium Model to Investigate Multicellular Dynamics During Angiogenesis

The development of biomimetic in vitro or ex vivo models that can incorporate the complexity of intact microvascular networks would enable the investigation of multicellular dynamics associated with angiogenesis and novel experimental platforms for pre‐clinical therapy testing. Recently, our laboratory has introduced the rat mesentery culture model as such a tool. The applicability of this model would be enhanced with the use of murine tissue, yet unfortunately, the mouse mesentery is avascular. A potential analog to the mesentery is the mesometrium, the connective tissue of the uterine horns. The objective of this study was to demonstrate that the mouse mesometrium contains intact microvascular networks that can be cultured to investigate multicellular dynamics during angiogenesis. Mesometrium windows, defined as the translucent connective tissues attached to the uterine horns, were harvested from adult C57Bl/6 female mice and cultured in minimum essential media with 20% fetal bovine serum for 3 and 5 days. The tissues were immunohistochemically labeled with platelet endothelial cell adhesion molecule (PECAM), neuron‐glial antigen 2 (NG2), and alpha‐smooth muscle actin (α‐SMA) to identify endothelial cells, pericytes, and smooth muscle cells, respectively. The mesometrium proved to have an intact microvascular network with arterioles, venules and capillaries. Arterioles and venules were differentiated by the PECAM‐positive cell morphology, and the α‐SMA‐positive cell morphology that wraps the vessels. α‐SMA and NG2 labeling confirmed the presence of smooth muscle cells along the larger vessels and pericytes along capillaries. After 5 days in culture, tissues remained viable as confirmed with a LIVE/DEAD assay. Compared to day 0, uncultured tissues, capillary sprouts per venule length was dramatically increased by day 3 and day 5 (Day 0: 0.09 ± 0.02; Day 3: 4.10 ± 0.75; Day 5: 4.37 ± 0.60 spouts/mm). For day 3 and day 5, capillary sprouting preferentially occurred of venules versus arterioles. Furthermore, while the number of capillary sprouts were comparable for day 3 and day 5, sprouts appeared to be longer on day 5. Capillary sprouts were also observed to contain NG2‐positive, and SMA‐negative pericytes. These results introduce the mouse mesometrium culture model as a novel ex vivo approach for elucidating pericyte‐endothelial cell dynamics associated with capillary sprouting at specific vessel locations in a real microvascular network scenario.Support or Funding InformationNIH R01AG049821, NIH P20GM103629

Peritoneal Washings After Power Morcellation in Laparoscopic Myomectomy: A Pilot Study

Study ObjectiveTo evaluate if peritoneal washings of the abdominopelvic cavity during laparoscopic myomectomy can detect leiomyoma cells after power morcellation. DesignProspective cohort pilot study. SettingUniversity of North Carolina Hospitals, an academic, tertiary referral center (Canadian Task Force classification II-2). PatientsPatients undergoing laparoscopic or robotic myomectomy for suspected benign leiomyoma by members of the Minimally Invasive Gynecologic Surgery division between September 2014 and January 2015. InterventionWashings of the peritoneal cavity were collected at 3 times during surgery: the beginning of the procedure once the peritoneal cavity was accessed laparoscopically, after the myoma was excised and myometrial incision closed, and after uncontained power morcellation. Measurements and Main ResultsTwenty patients were included in the analysis. The median morcellation time was 16 minutes (range, 2–36). The median specimen weight was 283.5 g (range, 13–935). Cytologic evaluation (ThinPrep with Papanicolaou staining) did not detect any smooth muscle cells. Cell block histology, however, detected spindle cells in 6 postmorcellation samples. Three of these 6 cases also had spindle cells detected on the postmyomectomy closure samples. When performed on the postmorcellation samples, desmin and smooth muscle actin immunostaining were positive, confirming the presence of smooth muscle cells. ConclusionCell block histology, but not cytology, can detect leiomyoma cells in peritoneal washings after power morcellation. With myomectomy, there is some tissue disruption that seems to cause cell spread even in the absence of morcellation. Further protocol testing might allow peritoneal washings to be used in assessing containment techniques and testing comparative safety of different morcellation methods.

Metallic zinc exhibits optimal biocompatibility for bioabsorbable endovascular stents.

Although corrosion resistant bare metal stents are considered generally effective, their permanent presence in a diseased artery is an increasingly recognized limitation due to the potential for long-term complications. We previously reported that metallic zinc exhibited an ideal biocorrosion rate within murine aortas, thus raising the possibility of zinc as a candidate base material for endovascular stenting applications. This study was undertaken to further assess the arterial biocompatibility of metallic zinc. Metallic zinc wires were punctured and advanced into the rat abdominal aorta lumen for up to 6.5months. This study demonstrated that metallic zinc did not provoke responses that often contribute to restenosis. Low cell densities and neointimal tissue thickness, along with tissue regeneration within the corroding implant, point to optimal biocompatibility of corroding zinc. Furthermore, the lack of progression in neointimal tissue thickness over 6.5months or the presence of smooth muscle cells near the zinc implant suggest that the products of zinc corrosion may suppress the activities of inflammatory and smooth muscle cells.

Development of small diameter nanofiber tissue engineered arterial grafts.

The surgical repair of heart and vascular disease often requires implanting synthetic grafts. While synthetic grafts have been successfully used for medium-to-large sized arteries, applications for small diameter arteries (<6 mm) is limited due to high rates of occlusion by thrombosis. Our objective was to develop a tissue engineered vascular graft (TEVG) for small diameter arteries. TEVGs composed of polylactic acid nanofibers with inner luminal diameter between 0.5 and 0.6 mm were surgically implanted as infra-renal aortic interposition conduits in 25 female C17SCID/bg mice. Twelve mice were given sham operations. Survival of mice with TEVG grafts was 91.6% at 12 months post-implantation (sham group: 83.3%). No instances of graft stenosis or aneurysmal dilatation were observed over 12 months post-implantation, assessed by Doppler ultrasound and microCT. Histologic analysis of explanted TEVG grafts showed presence of CD31-positive endothelial monolayer and F4/80-positive macrophages after 4, 8, and 12 months in vivo. Cells positive for α-smooth muscle actin were observed within TEVG, demonstrating presence of smooth muscle cells (SMCs). Neo-extracellular matrix consisting mostly of collagen types I and III were observed at 12 months post-implantation. PCR analysis supports histological observations. TEVG group showed significant increases in expressions of SMC marker, collagen-I and III, matrix metalloproteinases-2 and 9, and itgam (a macrophage marker), when compared to sham group. Overall, patency rates were excellent at 12 months after implantation, as structural integrity of these TEVG. Tissue analysis also demonstrated vessel remodeling by autologous cell.

Assessment of plaque composition by intravascular ultrasound and near-infrared spectroscopy: from PROSPECT I to PROSPECT II.

Atherosclerosis is the main cause of coronary artery disease (CAD), which is today the leading cause of death worldwide and will continue to be the first in the world in 2030. Vulnerable coronary plaques are usually characterized by a high content of necrotic core, a thin inflamed fibrous cap (intense accumulation of macrophages) and scarce presence of smooth muscle cells. None of these characteristics can be estimated by coronary angiography, which on the contrary underestimates the magnitude of atherosclerotic burden, particularly in earlier stage disease when positive vascular remodeling may allow "normal" lumen caliber despite substantial vascular wall plaque. The recognition of the ubiquity of substantial but non-flow limiting lesions that may be at high risk for subsequent plaque rupture has resulted in a paradigm shift in thinking about the pathophysiology of CAD, with the focus no longer solely on the degree of arterial luminal narrowing. This growing need for more information about coronary atherosclerosis in order to identify patients and lesions at risk for complications during PCI and for future adverse cardiac events has been the primary impetus for the development of novel intracoronary imaging methods able to detect plaque composition, in particular presence of a necrotic core/lipid pool, such as intravascular ultrasound virtual histology and near-infrared spectroscopy. These imaging technologies and their clinical and clinical/research applications are discussed in detail.

Angiogenic endothelial cell invasion into fibrin is stimulated by proliferating smooth muscle cells

These studies aimed to determine the effect of smooth muscle cells (SMCs) on angiogenic behavior of endothelial cells (ECs) within fibrin hydrogels, an extracellular matrix (ECM) commonly used in tissue engineering. We developed a 3-D, fibrin-based co-culture assay of angiogenesis consisting of aggregates of SMCs with ECs seeded onto the aggregates' surface. Using digital fluorescence micrography, EC matrix invasion was quantified by average length of sprouts (ALS) and density of sprout formation (DSF). We demonstrated that ECs and SMCs co-invade into the ECM in close proximity to one another. ECs that were co-cultured with SMCs demonstrated increased invasion compared to ECs that were cultured alone at all time points. At Day 19, the ALS of ECs in co-culture was 327+/−58μm versus 70+/−11μm of ECs cultured alone (p=.01). The DSF of co-cultured ECs was also significantly greater than that of ECs cultured alone (p=.007 on Day 19). This appeared to be a function of both increased EC invasion as well as improved persistence of EC sprout networks. At 7days, ECs in co-culture with proliferation-inhibited SMCs previously treated with Mitomycin-C (MMC) demonstrated significantly attenuated sprouting compared to ECs co-cultured with SMCs that were untreated with MMC (82+/−14μm versus 205+/−32μm; p<.05). In assays in which multiple co-culture aggregates were cultured within a single hydrogel, we observed directional invasion of sprouts preferentially towards the other aggregates within the hydrogel. In co-culture assays without early EC/SMC contact, the ALS of ECs cultured in the presence of SMCs was significantly greater than those cultured in the absence of SMCs by Day 3 (320+/−21μm versus 187+/−16μm; p<.005). We conclude that SMCs augment EC matrix invasion into 3-D fibrin hydrogels, at least in part resulting from SMC proliferative and invasive activities. Directed invasion between co-culture aggregates and augmented angiogenesis in the absence of early contact suggests a paracrine mechanism for the observed results.

Reconstruction of pulmonary artery with porcine small intestinal submucosa in a lamb surgical model: Viability and growth potential

This study investigated the time-dependent remodeling and growth potential of porcine small intestine submucosa as a biomaterial for the reconstruction of pulmonary arteries in a lamb model. Left pulmonary arteries were partially replaced with small intestine submucosal biomaterial in 6 lambs. Two animals each were humanely killed at 1, 3, and 6 months. Computed tomographic angiography, macroscopic examination of the implanted patch, and microscopic analysis of tissue explants were performed. All animals survived without complications. Patency and arborization of the pulmonary arteries were detected 6 months after implantation. There was no macroscopic narrowing or aneurysm formation in the patch area. The luminal appearance of the patch was similar to the intimal layer of the adjacent native pulmonary artery. Scanning electron microscopy showed that the luminal surface of the patch was covered by confluent cells. Immunohistochemical examination confirmed endothelialization of the luminal side of the patch in all of the explanted patches. The presence of smooth muscle cells in the medial layer was confirmed at all time points; however, expression of elastin, growth of the muscular layer, and complete degradation of patch material were detectable only after 6 months. The presence of c-Kit-positive cells suggests migration of multipotent cells into the patch, which may play a role in remodeling the small intestine submucosal biomaterial. Our data confirmed that remodeling and growth potential of the small intestine submucosal biomaterial are time dependent. Additional experiments are required to investigate the stability of the patch material over a longer period.

Abstract 521: The Thrombotic Potential of the Endothelium Is Regulated by Hemodynamic Flow

BACKGROUND: Vascular wall mechanisms underlying human arterial and venous thrombosis are still evolving. TNFα and other cytokines have been shown to be potent mediators of endothelial cell-derived tissue factor in vitro, which mediates fibrin deposition in the vascular wall. Circulating levels of TNFα detected in humans with cardiovascular disease are approximately 0.05 ng/ml. METHODS: Human endothelial cells were co-cultured with or without smooth muscle cells in the presence or absence of human-derived, region-specific hemodynamics. Endothelial cells were exposed to TNFα at various concentrations and incubated in human, platelet-free-plasma supplemented by Alexa 488-labeled fibrinogen. Conversion of A488-fibrinogen to A488-fibrin and deposition on the endothelium was quantified by confocal microscopy. RESULTS: In static cultures, samples treated with 1ng/ml TNFα demonstrated minimal fibrin deposition whereas samples treated with 10ng/ml or 20ng/ml exhibited dense fibrin networks (1.17e5 vs. 2.69e7 vs. 3.61e7 mean fluorescence intensity, respectively). Fibrin deposition was tissue factor-dependent. In contrast, endothelial/smooth muscle cell co-cultures primed with inflammatory-prone hemodynamics derived from the internal carotid sinus and 0.05ng/ml TNFα deposited a dense fibrin network (1.9e7 mean fluorescence intensity). Identical experiments in which only endothelial cells were cultured yielded similar results demonstrating the result is hemodynamic-specific and not a consequence of the presence of smooth muscle cells. CONCLUSIONS: Hemodynamic priming of endothelial cells in vitro shifts the dose-dependent fibrin deposition response to TNFα into a more human physiological range, two orders of magnitude below that required to induce fibrin deposition in static endothelial cell cultures. Similar approaches can be used to investigate the differential mechanisms underlying vascular wall-mediated venous and arterial thrombosis.

Sphingosine 1-phosphate-induced release of TIMP-2 from vascular smooth muscle cells inhibits angiogenesis.

Following myocardial infarction, angiogenesis occurs as a result of thrombus formation, which permits reperfusion of damaged myocardium. Sphingosine 1-phosphate (S1P) is a naturally occurring lipid mediator released from platelets and is found in high concentrations at sites of thrombosis. S1P might therefore be involved in regulating angiogenesis following myocardial infarction and might influence reperfusion. The aims of this study were to determine the effects of S1P in human coronary arterial cell angiogenesis and delineate the subsequent mechanisms. An in vitro model of angiogenesis was developed using a co-culture of human coronary artery endothelial cells, human coronary smooth muscle cells and human fibroblasts. In this model, S1P inhibited angiogenesis and this was dependent on the presence of smooth muscle cells. The mechanism of the inhibitory effect was through S1P-induced release of a soluble mediator from smooth muscle cells. This mediator was identified as tissue inhibitor of metalloproteinase-2 (TIMP-2). Release of TIMP-2 was dependent on S1P-induced activation of Rho kinase and directly contributed to incomplete formation of endothelial cell adherens junctions. This was observed as a diffuse localisation of VE-cadherin, leading to decreased tubulogenesis. A similar inhibitory response to S1P was demonstrated in an ex vivo human arterial model of angiogenesis. In summary, S1P-induced inhibition of angiogenesis in human artery endothelial cells is mediated by TIMP-2 from vascular smooth muscle cells. This reduces the integrity of intercellular junctions between nascent endothelial cells. S1P might therefore inhibit the angiogenic response following myocardial infarction.

Development of Apolipoprotein E–Deficient Mice

The first mouse carrying a deletion mutation in its apolipoprotein E (apoE) gene was born in our mouse colony in the summer of 1991. Our ongoing experiments to that point had indicated that this would happen, but the realization that the mouse in front of my eyes had inherited a mutation that we had designed and made in the laboratory filled me with awe. It did not occur to me immediately that this mouse would develop hypercholesterolemia and atherosclerosis and dictate the direction of my research for the next 20 years. I here briefly describe the background and my personal experience that led to the development of apoE-deficient mice as a model for human atherosclerosis. As a research scientist in the Department of Genetics at the University of Wisconsin, my main interest during the early 1980s was directed toward understanding how different types of DNA recombination contribute to the architecture and diversity of individual genomes within human populations and to DNA differences between humans and nonhuman primates. My studies clearly showed that new alleles and new genes are constantly being formed in the human genome by recombinational events, particularly between existing members of multigene families. There also appeared to be hot spots for recombination. By tracing a history of the human haptoglobin-gene cluster from the present-day structures in human populations and in other primates, I found that a single recombination event between related genes often produces much more drastic changes in the genome than those that result from simple point mutations.1 Whether this frequent yet natural occurrence of homologous recombination in the mammalian genome could be used to alter the genome of mammalian cells in culture in a preplanned fashion became a question of interest to several laboratories.2,3 An affirmative answer was first obtained in 1985, …