Medial Smooth Muscle Cells Research Articles

Temporal network based analysis of cell specific vein graft transcriptome defines key pathways and hub genes in implantation injury.

Vein graft failure occurs between 1 and 6 months after implantation due to obstructive intimal hyperplasia, related in part to implantation injury. The cell-specific and temporal response of the transcriptome to vein graft implantation injury was determined by transcriptional profiling of laser capture microdissected endothelial cells (EC) and medial smooth muscle cells (SMC) from canine vein grafts, 2 hours (H) to 30 days (D) following surgery. Our results demonstrate a robust genomic response beginning at 2 H, peaking at 12–24 H, declining by 7 D, and resolving by 30 D. Gene ontology and pathway analyses of differentially expressed genes indicated that implantation injury affects inflammatory and immune responses, apoptosis, mitosis, and extracellular matrix reorganization in both cell types. Through backpropagation an integrated network was built, starting with genes differentially expressed at 30 D, followed by adding upstream interactive genes from each prior time-point. This identified significant enrichment of IL-6, IL-8, NF-κB, dendritic cell maturation, glucocorticoid receptor, and Triggering Receptor Expressed on Myeloid Cells (TREM-1) signaling, as well as PPARα activation pathways in graft EC and SMC. Interactive network-based analyses identified IL-6, IL-8, IL-1α, and Insulin Receptor (INSR) as focus hub genes within these pathways. Real-time PCR was used for the validation of two of these genes: IL-6 and IL-8, in addition to Collagen 11A1 (COL11A1), a cornerstone of the backpropagation. In conclusion, these results establish causality relationships clarifying the pathogenesis of vein graft implantation injury, and identifying novel targets for its prevention.

Local Medial Microenvironment Directs Phenotypic Modulation of Smooth Muscle Cells After Experimental Renal Transplantation

Smooth muscle cells (SMCs) play a key role in the pathogenesis of occlusive vascular diseases, including transplant vasculopathy. Neointimal SMCs in experimental renal transplant vasculopathy are graft-derived. We propose that neointimal SMCs in renal allografts are derived from the vascular media resulting from a transplantation-induced phenotypic switch. We examined the molecular changes in the medial microenvironment that lead to phenotypic modulation of SMCs in rat renal allograft arteries with neointimal lesions. Dark Agouti donor kidneys were transplanted into Wistar Furth recipients and recovered at day 56. Neointimal and medial layers were isolated using laser microdissection. Gene expression was analyzed using low-density arrays and confirmed by immunostaining. In allografts, neointimal SMCs expressed increased levels of Tgf β1 and Pdgfb. In medial allograft SMCs, gene expression of Ctgf, Tgf β1 and Pdgfrb was upregulated. Gene expression of Klf4 was upregulated as well, while expression of Sm22α was downregulated. Finally, PDGF-BB-stimulated phenotypically modulated SMCs, as evidenced by reduced contractile function in vitro which was accompanied by increased Klf4 and Col1α1, and reduced α-Sma and Sm22α expression. In transplant vasculopathy, neointimal PDGF-BB induces phenotypic modulation of medial SMCs, through upregulation of KLF4 in the media to contribute to (further) expansion of the neointima.

Comparison of the SYNERGY with the PROMUS (XIENCE V) and bare metal and polymer-only Element control stents in porcine coronary arteries

This study evaluated vascular compatibility of the novel platinum chromium alloy Element stent platform delivering abluminal everolimus from a poly-lactide-co-glycolide bioabsorbable polymer (SYNERGY stent), currently undergoing clinical trial, compared with the PROMUS (XIENCE V) and bare metal and polymer-only Element stents. Stents (n=161) were implanted one per coronary artery in 72 swine at a stent-to-artery ratio of 1.1:1. Similar numbers of each device group were explanted at each of 30, 90, 180, and 360 days (except no PROMUS (XIENCE V) stent at 360 days) for pathological analysis. There was no stent thrombosis, myocardial infarction, or strut fractures in any group. Vascular response was similar between the SYNERGY and PROMUS (XIENCE V) stents, with no thrombi and complete endothelialisation on both scanning electron microscopy and histology at 30, 90 and 180 days. There were no significant differences for the morphologic parameters of luminal thrombus, endothelial cell coverage, strut tissue coverage, inflammation, internal elastic lamina (IEL) disruption, external elastic lamina (EEL) disruption and medial smooth muscle cell loss across device groups or between time points, but there was mild but greater (p<0.0001) para-strut fibrin at 30 days for both drug-eluting stents (DES) compared with the bare and polymer-only controls; this difference completely dissipated by 90 days. Inflammation was predominantly minimal to mild for all device types. No morphometric parameters, including intimal thickness, stent profile-based area stenosis, and EEL area were significantly different when comparing the SYNERGY stent with the bare metal Element and polymer-only Element control stents at 90, 180 and 360 days. In this non-injured porcine coronary artery model, the bioabsorbable polymer SYNERGY stent demonstrated vascular compatibility equivalent to the PROMUS (XIENCE V) stent and to the bare metal and polymer-only Element stents.

Monitoring the Inflammatory Process by Surface Enhanced Nanoimaging Microscopy

The scope of this study highlights application of an emerging hybrid vibrational nanoimaging techniques based on surface enhanced nanoimaging microscopy in investigating the inflammatory process in the vascular system and neurodegenerative disorder. The experiments were performed on transgenic rat and ApoE knockout mice model, intravenously injected with USPIO based MR contrast agent. Marked signal enhancement were observed in contrast treated specimens in specific pathological regions, which is known to be infiltrated by helper T cells. The findings were subsequently correlated with the results from MR imaging and immunohistochemistry. The focus of this work encompasses the latest innovation of nanoimaging technology in monitoring the inflammatory process in neurodegenerative diseases, and implementing the first observation of intracellular iron uptakes in the endothelial and medial smooth muscle cells based on Coherent anti-Stokes Raman Scattering (SECARS) Microscopy.

The double‐edged sword of fibronectin in atherosclerosis

Atherosclerosis, the leading cause of death in Westernized countries, derives from a non‐resolving inflammation of the large arteries. A critical step in the initiation of an atherosclerotic plaque is the expansion of intimal space (the area between the endothelial cell (EC) lining and the medial smooth muscle cell layer) in susceptible regions of arteries (Tabas et al, 2007). The disease‐prone locations are largely determined by arterial blood flow characteristics, with branch points and the lesser curvatures of arteries, where the flow is turbulent, being particularly disposed to plaque formation. At these sites, the normally thin intimal space expands with the deposition of extracellular matrix (ECM), which then serves as a sticky surface to trap LDL particles that are constantly crossing the endothelial layer to enter the artery. One reason for this retention is that the apoB100 molecule on the LDL particle has a specific domain that binds tightly to the ECM, and genetic causation studies in a mouse model of atherosclerosis have shown that when this domain is deleted, plaque burden is significantly reduced despite equivalent hyperlipidemia (Skalen et al, 2002). As implied by the ‘Response to Retention’ hypothesis (Tabas et al, 2007), the retained lipoprotein particles set in motion the invasion of the arterial wall by monocytes that quickly become macrophages that engorge themselves on cholesterol and the other lipids to form foam cells. Foam cells not only physically expand the plaque, but by secreting a variety of chemokines and cytokines, they also create and amplify an inflammatory state (Fig 1). Figure 1. Plasma derived FN promotes atherogenesis in mouse models of atherosclerosis. pFN, synthesized in the liver, is deposited in susceptible regions of the arteries and contributes to early atherosclerotic lesion formation and the recruitment of smooth muscle cells that shape the fibrous cap of advanced lesions (upper panel). …

Type I immune response cytokine–chemokine cascade is associated with pulmonary arterial hypertension

Perivascular infiltrating mononuclear cells have been described in the vasculopathy found in multiple types of pulmonary arterial hypertension (PAH). We determined the expression of a specific type 1 immune response cytokine-chemokine cascade-interleukin (IL)-18 → (monokine induced by γ-interferon [MIG]/chemokine [C-X-C motif] ligand [CXCL] 9, interferon γ-induced protein [IP]-10/CXCL10 and interferon-inducible T-cell α chemoattractant [ITAC]/CXCL11)-in plasma samples from individuals with World Health Organization (WHO) Group 1 PAH. We analyzed cytokine and chemokine protein levels in plasma from 43 individuals with WHO Group 1 PAH by enzyme-linked immunosorbent assay compared with 35 healthy individuals. Immunohistochemical studies on tissue specimens from WHO Group 1 PAH patients were performed for cytokines and chemokines and their respective receptors. Plasma IL-18 levels from WHO Group 1 PAH patients were significantly increased compared with healthy controls. Downstream chemokine CXCL10, but not CXCL9 or CXCL11, was markedly elevated compared with controls. Cellular sources of IL-18 were medial but not intimal smooth muscle cells. IL-18Rα was expressed from medial smooth muscle cells, endothelial cells, and mononuclear cells. CXCL10 and its main receptor, CXCR3, were expressed from infiltrating vascular wall mononuclear cells. These data suggest that augmented expression of IL-18 and CXCL10 may perpetuate an inflammatory milieu that eventually contributes to the vascular obstruction characteristic of PAH.

Serine protease inhibitor A3 in atherosclerosis and aneurysm disease

Remodeling of extracellular matrix (ECM) plays an important role in both atherosclerosis and aneurysm disease. Serine protease inhibitor A3 (serpinA3) is an inhibitor of several proteases such as elastase, cathepsin G and chymase derived from mast cells and neutrophils. In this study, we investigated the putative role of serpinA3 in atherosclerosis and aneurysm formation. SerpinA3 was expressed in endothelial cells and medial smooth muscle cells in human atherosclerotic lesions and a 14-fold increased expression of serpinA3n mRNA was found in lesions from Apoe-/- mice compared to lesion-free vessels. In contrast, decreased mRNA expression (-80%) of serpinA3 was found in biopsies of human abdominal aortic aneurysm (AAA) compared to non-dilated aortas. Overexpression of serpinA3n in transgenic mice did not influence the development of atherosclerosis or CaCl2-induced aneurysm formation. In situ zymography analysis showed that the transgenic mice had lower cathepsin G and elastase activity, and more elastin in the aortas compared to wild-type mice, which could indicate a more stable aortic phenotype. Differential vascular expression of serpinA3 is clearly associated with human atherosclerosis and AAA but serpinA3 had no major effect on experimentally induced atherosclerosis or AAA development in mouse. However, serpinA3 may be involved in a phenotypic stabilization of the aorta.

Cyclosporine immunosuppression does not prevent the production of donor-specific antibody capable of mediating allograft vasculopathy

Late cardiac graft rejection, primarily mediated by allograft vasculopathy (AV), remains a major limitation to cardiac transplantation, even in the face of significant calcineurin inhibitor (CNI) immunosuppression. The role played by alloantibody in AV is unclear. Evidence that CNI immunosuppression suppresses CD4(+) T-cell function would suggest that antibody production and effector function would be severely limited in CNI-treated patients. In this study we examine the capacity of CNI-treated animals to develop effective alloantibody that can mediate AV. Wild-type (WT) B6 mice were alloimmunized using donor splenocytes or a fully major histocompatibility complex-mismatched allogeneic abdominal aortic graft in the presence of CNI immunosuppression (30 or 50 mg/kg/day cyclosporine A). Anti-serum was harvested and tested using complement-dependent in vitro cytotoxicity assays. Anti-serum was passively transferred to immunodeficient RAG1(-/-) recipients of allogeneic grafts. C4d deposition was quantified in the allografts from WT recipients. CNI immunosuppression did not prevent the development of alloantibody in response to either immunization method (p < 0.05). Passive transfer of anti-serum generated AV lesions in immunodeficient graft recipients and mediated complement-dependent destruction of donor cells (p < 0.05). C4d deposition was localized to the media of grafts of CNI treated animals. CNI therapy does not prevent the production of alloantibody with the capacity to mediate AV. C4d deposition in the media suggests a role for medial smooth muscle cell loss in antibody-mediated AV lesion development in our model.

Abstract 29: Fate-Tracing Demonstrates that Medial SMCs, but Not Mature ECs, Contribute to Neointimal Cell Population in Injured Arteries

It has been proposed that various groups of cells, such as medial SMCs (mSMCs), bone marrow progenitors, and mature ECs (mECs) hold the potential to become neointimal SMCs via phenotypic switching, differentiation, or endothelial-mesenchymal-transition (EMT). However, no definitive evidence has been provided to support these claims, which renders the in vivo significance of these events in question. Using an inducible Cre-loxP system, where the production of a reporter (e.g. β-gal) is driven by a highly definitive marker gene for mSMCs (e.g. SMC myosin heavy chain, SMMHC) or mECs (e.g. stem cell leukemia, SCL), we traced the fate of these cells during neointimal hyperplasia (NIH) in ligated common carotid and wire-injured femoral arteries. Tamoxifen induction resulted in a homogeneous labeling of mSMCs of the unmanipulated arteries of R26R;SMMHC-CreERtm mice. Following arterial injury, SMCs that were initially located in the medial layer presented in neointimal lesions; however, the fraction of these cells varied widely among vessels with either type of injury. This heterogeneity was not secondary to the random loss of the genetic label as evidenced by the stable β-gal activity in SMC culture. Addition of a second (4wks after surgery) induction did not change the pattern and fraction of the labeled SMCs in the neointima, indicating that few recruited progenitors transform into mature SMCs. This observation is further supported through examination of mice chimeric for R26R;SMMHC-CreERtm bone marrow, where β-gal activity was not detected in injured arteries though strongly induced in culture following PDGF treatment. Neointima formed in R26R;SCL-CreERtm arteries demonstrated a co-localization of β-gal activity to the repopulated ECs, with none being detected in cells underneath the EC monolayer, suggesting a role for mECs in re-endothelialization and indicating EMT as a rare event in NIH development. We have provided direct evidence for the contribution of mSMCs to NIH. The recruited vascular progenitors, particularly bone marrow cells, do not differentiate into fully mature SMCs in NIH lesion. EMT does not serve as a mechanism for populating neointimal cells following vascular injury while occurring under other vascular disease conditions.

Abstract 102: Model-Dependent Influence of Prior Splenectomy on Experimental Abdominal Aortic Aneurysm Progression

Objective: Abdominal aortic aneurysm (AAA) is a macrophage-driven, inflammatory vascular disease. Thus, mobilization of macrophage precursors from the bone marrow and/or splenic reservoir may represent a crucial initial step for AAA formation. Prior evidence suggests that splenic monocytes are important initial contributors to ischemic myocardial injury, their role in AAA pathogenesis remains unknown. We surgically removed the spleen prior to AAA creation to evaluate the influence of splenic monocytes on aneurysm pathophysiology. Methods: AAAs were created in either 1) male C57BL/6 mice by infrarenal intra-aortic porcine pancreatic elastase (PPE) infusion or 2) male ApoE deficient mice by continuous subdermal angiotensin II infusion (ApoE KO/Ang II model). Splenectomy was performed immediately prior to AAA creation in both models. Aortic diameter was serially monitored via ultrasonography, and aortae harvested at sacrifice for histopathology. Results: In sham splenectomy mice, PPE infusion led to significant aortic enlargement. AAA (≥50% aortic diameter increase) developed in all sham/PPE mice within 14 days (n=10). Splenectomy effectively eliminated PPE-induced aortic enlargement, with preservation of medial elastin and smooth muscle cell density and minimal inflammation compared to sham. In contrast, Ang II infusion in ApoE deficient produced similar-sized or larger AAA in splenectomized mice compared to sham, with elastin degradation, smooth muscle cell depletion undistinguishable between the groups. Conclusion: Splenectomy confers differential protection from AAA in mice, depending on the model/method employed for aneurysm formation. While this experiment underscores important limitations to mouse-based AAA modeling, the splenic monocyte reservoir may represent an important driver of AAA pathophysiology.

Abstract 427: Transforming Growth Factor β/Smad3 Stimulates Smooth Muscle Cell Migration and Proliferation Through a Signaling Pathway that Involves C-X-C Chemokine Receptor Type 4 Expression and Activation of Mitogen-Activated Protein Kinase

Introduction: Intimal hyperplasia is a complex process that requires vascular smooth muscle cell (SMC) migration and proliferation. We have previously shown that levels of transforming growth factor beta (TGF-β) and its signaling protein Smad3 are elevated following arterial injury and contribute to intimal hyperplasia. In this study we exam whether these effects might be mediated through a mechanism that involves the chemokine stromal cell derived factor (SDF-1α) and its receptor C-X-C receptor type 4 (CXCR4). Methods and Results: A microarray was used to better understand the signaling pathways through which TGF-β and Smad3 stimulate SMC proliferation and migration. Overexpression of Smad3 in SMCs followed by stimulation with TGF-β (5 ng/ml) for 6 hours resulted in significant alteration of ∼300 genes, the most dramatic effect being a &gt;500 fold upregulation of CXCR4. Quantitative RT-PCR and Western blotting confirmed that Smad3/TGF- β increased CXCR4 transcription and protein expression by 155±9 and 24±5 -fold, respectively. Immunocytochemistry and flowcytometry analysis revealed a 6-fold increase in CXCR4 positive cells in TGF-β stimulated Smad3 overexpressing SMCs compared to control (P&lt;0.05). TGF-β/Smad3 treated SMCs exhibited enhanced migration (4-fold VS control, P&lt;0.01) in response to SDF-1α (100 ng/ml) which was eliminated after preincubation of SMCs with CXCR4 inhibitor AMD3100 (10 μM). Moreover, there was a 45±9% increase in proliferation (BrdU) in response to SDF-1α in TGF-β/Smad3 treated SMCs compared to GFP control (P&lt;0.05). To explore the mechanism by which SDF-1α/CXCR4 produces proliferation and migration in SMCs, we studied the intracelluar signaling protein mitogen-activated protein kinase (MAPK), which is a known intermediate for both processes. Incubation of TGF-β/Smad3 SMCs with SDF-1α led to activation of MAPK (11-fold) as well as nuclear translocation of CXCR4 in a dose and time dependent manner. Conclusions: Our data demonstrate that TGF-β through Smad3 stimulates expression of CXCR4 in medial vascular SMCs. These SMCs in response to SDF-1α then proliferate and migrate to the subintimal layer. These findings suggest a novel pathway through which TGF-β enhances intimal hyperplasia at sites of arterial injury.

Abstract 519: Calmodulin Is a Marker of a Distinct Smooth Muscle Cell Population Recruited by Plaque-Derived Macrophages from the Human Carotid Artery Media

Background: Different experimental observations suggest the expansion of a distinct medial smooth muscle cell (SMC) subset during atheromatous plaque establishment and restenosis. Objectives: We investigated the presence and features of such SMC subset in cell cultures derived from human carotid endarterectomy (CEA) specimens; moreover, we aimed at finding markers that could play a role in SMC behavior. Methods and Results: CEA specimens comprised a grossly undiseased (UP) and a diseased portion (DP). The UP contained a thin intimal thickening with the underlying media and the DP an atherosclerotic plaque with the underlying media. Cell cultures were initiated by tissue explantation. From plaque tissues only macrophage-derived foam cells were retrieved. From medial tissues 2 distinct SMC phenotypes were identified: 1) large SMCs, flat with a monolayered growth pattern, isolated from the UP media; 2) small SMCs, fusiform and growing in multilayers, isolated from both the UP and DP media only when cocultured with plaque-derived macrophages. Small SMCs displayed higher proliferative and migratory activities and a poor level of differentiation compared with large SMCs. Two-dimensional polyacrylamide gel electrophoresis followed by tandem mass spectrometry showed that calmodulin (CaM), a calcium-binding protein involved in cell-cycle regulation, was mainly expressed in small SMCs. This result was confirmed using a specific CaM antibody. Coculture of large SMCs with plaque-derived macrophages induced a switch to the small phenotype and was associated with increased CaM expression. Consistently, the specific CaM inhibitor W-7 hampered the large-to-small switch and predominantly inhibited the proliferation of small SMCs. In vivo, CaM was markedly expressed in atherosclerotic plaques whereas it was barely detectable in the media. Conclusions: Plaque-derived macrophages promote the selective migration of a distinct SMC subpopulation exhibiting features of an “atheroma-prone” phenotype. CaM is a marker of small SMCs in vitro and of plaque SMCs in vivo, and plays a role in SMC phenotypic modulation. Further studies on these SMC populations can be instrumental in understanding and influencing the evolution of atherosclerosis.

Abstract 176: Extracellular S100A4 Is a Key Modulator of Arterial Smooth Muscle Cell Phenotypic Transition

Background: It has been proposed that smooth muscle cells (SMCs) from the arterial wall are heterogeneous and that only a subset of medial SMCs are prone to accumulate into the intima leading to atheromatous plaque formation. We isolated 2 distinct SMC phenotypes from porcine coronary artery: spindle-shaped (S) and rhomboid (R). Biological features of R-SMCs (i.e. enhanced proliferative and migratory activities as well as poor level of differentiation) explain their capacity to accumulate into the intima. We identified S100A4 as a marker of R-SMCs in vitro and of intimal SMCs both in pig and human. S100A4 is an intracellular Ca2+ binding protein that can also be secreted; it has extracellular functions probably via the receptor for advanced glycation end products (RAGE). Objective: Investigate the role of S100A4 in SMC phenotypic change, a process characteristic of atherosclerotic plaque formation. Methods and results: Transfection of a human S100A4-containing plasmid in S-SMCs (practically devoid of S100A4) led to overexpression of S100A4 in approximately 10% of SMCs, release of S100A4, and a transition towards a R-phenotype of the whole SMC population. Moreover, treatment of S-SMCs with S100A4-rich conditioned medium collected from S100A4-transfected S-SMCs induced a transition towards a R-phenotype, which was associated with increased proliferative and migratory activities as well as downregulation of SMC differentiation markers (i.e. alpha-smooth muscle actin and smooth muscle myosin heavy chains). It yielded activation of NF-kappa B without changing RAGE mRNA baseline level. Blockade of extracellular S100A4 in R-SMCs with S100A4 neutralizing antibody induced a transition from R- to S-phenotype, decreased proliferative activity and upregulated SMC differentiation markers. In contrast, silencing of S100A4 mRNA in R-SMCs and during platelet-derived growth factor-BB-induced S-to-R-phenotypic change did not affect SMC morphology in spite of decreased proliferative activity. Conclusions: Our results show that extracellular S100A4 modulates SMC phenotypic changes and is essential for the establishment of the R-phenotype. It could be a new target to prevent SMC accumulation during atherosclerosis and restenosis.

Growth-associated hyperphosphatemia in young recipients accelerates aortic allograft calcification in a rat model

Cardiovascular allografts in the young have limited durability because of early graft calcification. The objective of this study was to examine the hypothesis that growth-associated hyperphosphatemia in youth accelerates aortic allograft calcification by osteogenic transformation of graft medial smooth muscle cells (SMCs). The descending aortas of donor rats were subcutaneously transplanted into recipients. Syngeneic (Lewis-to-Lewis) transplantations between 3-week-old "young" (Y) rats and between 10-week-old "adult" (A) rats were combined with standard (ST, 0.9% phosphate) and low-phosphate (LP, 0.2%) diets, resulting in Y-ST, Y-LP, and A-ST groups. Allotransplantations (Brown-Norway-to-Lewis) involving these ages and diets were also made. The grafts and sera were retrieved from recipients after 14 days. Cultured rat aortic SMCs were used to analyze the effects of tumor necrosis factor-alpha (TNF-α) and phosphate on SMC calcification. In vivo, serum phosphate levels were higher in Y-ST (11.5 mg/dL) than those in Y-LP (8.9 mg/dL) and A-ST (8.5 mg/dL). Graft medial calcification appeared severe only in Y-ST. Allotransplants did not affect these outcomes. Graft medial cells showed phenotypic changes (contractile to synthetic) and osteogenic transformation (α-smooth muscle actin to Runx2 and osteocalcin), together with up-regulated proinflammatory TNF-α and sodium-phosphate cotransporter, Pit-1, despite ages and diets. In vitro, TNF-α induced phenotypic changes and osteogenic transformation of SMCs with Pit-1 up-regulation, but SMC calcification occurred only with high phosphate (4.5 mmol/L). Growth-associated hyperphosphatemia with inflammatory responses may be essential for accelerating allograft calcification in youth and could be a therapeutic target.

Changing topographic Hox expression in blood vessels results in regionally distinct vessel wall remodeling

SummaryThe distinct topographic Hox expression patterns observed in vascular smooth muscle cells (VSMCs) of the adult cardiovascular system suggest that these transcriptional regulators are critical for maintaining region-specific physiological properties of blood vessels. To test this proposition, we expanded the vascular Hoxc11 expression domain normally restricted to the lower limbs by utilizing an innovative integrated tetracycline regulatory system and Transgelin promoter elements to induce Hoxc11 expression universally in VSMCs of transgenic mice. Ectopic Hoxc11 expression in carotid arteries, aortic arch and descending aorta resulted in drastic vessel wall remodeling involving elastic laminae fragmentation, medial smooth muscle cell loss, and intimal lesion formation. None of these alterations were observed upon induction of Hoxc11 transgene expression in the femoral artery, i.e. the natural Hoxc11 activity domain, although this vessel was greatly enlarged, comparable to the topographically restricted vascular changes seen in Hoxc11−/− mice. To begin defining Hoxc11-controlled pathways of vascular remodeling, we performed immunolabeling studies in conjunction with co-transfection and chromatin immunoprecipitation (ChIP) assays using mouse vascular smooth muscle (MOVAS) cells. The results suggest direct transcriptional control of two members of the matrix metalloproteinase (Mmp) family, including Mmp2 and Mmp9 that are known as key players in the inception and progression of vascular remodeling events. In summary, the severe vascular abnormalities resulting from the induced dysregulated expression of a Hox gene with regional vascular patterning functions suggest that proper Hox function and regulation is critical for maintaining vascular functional integrity.

A20-Mediated Modulation of Inflammatory and Immune Responses in Aortic Allografts and Development of Transplant Arteriosclerosis

Transplant arteriosclerosis (TA) is the pathognomonic feature of chronic rejection, the primary cause of allograft failure. We have shown that the NF-κB inhibitory protein A20 exerts vasculoprotective effects in endothelial and smooth muscle cells (SMC), and hence is a candidate to prevent TA. We sought direct proof for this hypothesis. Fully mismatched, C57BL/6 (H2) into BALB/c (H2), aorta to carotid allografts were preperfused with saline, recombinant A20 adenovirus (rAd.A20) or rAd.β-galactosidase (β-gal), implanted, harvested 4 weeks after transplantation, and analyzed by histology, immunohistochemistry, and immunofluorescence staining. We measured indoleamine 2,3-dioxygenase, interleukin-6, and transforming growth factor-β mRNA and protein levels in nontransduced, and rAd.A20 or rAd.β-gal-transduced human SMC cultures after cytokine treatment. Vascular overexpression of A20 significantly reduced TA lesions. This correlated with decreased graft inflammation and increased apoptosis of neointimal SMC. Paradoxically, T-cell infiltrates increased in A20-expressing allografts, including the immunoprivileged media, which related to A20 preventing indoleamine 2,3-dioxygenase upregulation in SMC. However, infiltrating T cells were predominantly T-regulatory cells (CD25+/Forkhead Box P3 [FoxP3+]). This agrees with A20 inhibiting interleukin-6 and promoting transforming growth factor-β production by medial SMC and in SMC cultures exposed to cytokines, which favors differentiation of regulatory over pathogenic T cells. In summary, A20 prevents immune-mediated remodeling of vascular allografts, therefore reduces TA lesions by affecting apoptotic and inflammatory signals and modifying the local cytokine milieu to promote an immunoregulatory response within the vessel wall. This highlights a novel function for A20 in local immunosurveillance, which added to its vasculoprotective effects, supports its therapeutic promise in TA.

Contribution of monocyte-derived macrophages and smooth muscle cells to arterial foam cell formation

Smooth muscle cells (SMCs) are the main cell type in intimal thickenings and some stages of human atherosclerosis. Like monocyte-derived macrophages, SMCs accumulate excess lipids and contribute to the total intimal foam cell population. In contrast, apolipoprotein (Apo)E-deficient and LDL receptor-deficient mice develop atherosclerotic lesions that are macrophage- as opposed to SMC-rich. The lesser contribution of SMCs to lesion development in these mouse models has distracted attention away from the importance of SMC cholesterol homeostasis in the artery wall. Intimal SMCs accumulate excess amounts of cholesteryl esters when compared with medial layer SMCs, possibly explained by reduced ATP-binding cassette transporter A1 expression and ApoA-I binding to intimal-type SMCs. The aim of this review is to compare the relative contribution of monocyte-derived macrophages and SMCs to human vs. mouse atherosclerosis, and describe what is known about lipid uptake and removal mechanisms contributing to arterial macrophage and SMC foam cell formation. An increased understanding of the contribution of these cell types to lesion development will help to delineate their relative importance in atherogenesis and as potential therapeutic targets.

Human obesity and endothelium‐dependent responsiveness

Obesity is an ongoing worldwide epidemic. Besides being a medical condition in itself, obesity dramatically increases the risk of development of metabolic and cardiovascular disease. This risk appears to stem from multiple abnormalities in adipose tissue function leading to a chronic inflammatory state and to dysregulation of the endocrine and paracrine actions of adipocyte-derived factors. These, in turn, disrupt vascular homeostasis by causing an imbalance between the NO pathway and the endothelin 1 system, with impaired insulin-stimulated endothelium-dependent vasodilation. Importantly, emerging evidence suggests that the vascular dysfunction of obesity is not just limited to the endothelium, but also involves the other layers of the vessel wall. In particular, obesity-related changes in medial smooth muscle cells seem to disrupt the physiological facilitatory action of insulin on the responsiveness to vasodilator stimuli, whereas the adventitia and perivascular fat appear to be a source of pro-inflammatory and vasoactive factors that may contribute to endothelial and smooth muscle cell dysfunction, and to the pathogenesis of vascular disease. While obesity-induced vascular dysfunction appears to be reversible, at least in part, with weight control strategies, these have not proved sufficient to prevent the metabolic and cardiovascular complication of obesity on a large scale. While a number of currently available drugs have shown potentially beneficial vascular effects in patients with obesity and the metabolic syndrome, elucidation of the pathophysiological mechanisms underlying vascular damage in obese patients is necessary to identify additional pharmacologic targets to prevent the cardiovascular complications of obesity, and their human and economic costs. This article is part of a themed section on Fat and Vascular Responsiveness. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-3.

Acute Regeneration and Chronic Acellular Transformation of Rabbit Cryopreserved Aortic Allografts

An analysis of rabbit cryopreserved aortic allografts excised on postoperative days (POD) 2, 5, 11, 60, 210, 360, and 720, as well as controls that were untransplanted native aortas and cryopreserved aortas, was performed. On POD2, the number of medial smooth muscle cells in the allografts was reduced to approximately 50%. Ki-67 analysis revealed that medial smooth muscle cells in the allografts proliferated from the 2nd day. By the 11th day, their proliferation ceased and the number of medial smooth muscle cells was restored to almost at the same level as in the controls. Polymorphic microsatellite DNA marker analysis disclosed that the restored medial smooth muscle cells were of donor origin. From 7 months through 2 years, the media of cryopreserved aortic allografts were transformed into acellular structures, in which the elastic fibers were preserved. On the other hand, newly accumulated smooth muscle cells were observed in the adventitia just outside of acellular media after 7 months. In some cases, scattered lamellar calcium deposition was observed in the same regions. This study presents a comprehensive documentation of regeneration and acellular transformation in cryopreserved aortic allografts based on short and long-term analysis.

Age-dependent ultrastructural changes of coronary artery in spontaneously hypertensive rats

The age-dependent differences in basic cardiovascular parameters, geometry and structure of coronary arteries between Wistar and spontaneously hypertensive rats (SHR) were evaluated. SHR of the age 3-, 9-, 17-, and 52-week and age-matched Wistar rats were used. Blood pressure (BP) was measured by the plethysmographic method. Animals were perfused with a glutaraldehyde fixative under pressure of 90 mmHg (3-week-old) and 120 mmHg (9-, 17-, 52-week-old). Coronary arteries were processed for electron microscopy. The proportions and cross sectional areas (CSA) of extracellular matrix in intima and media, endothelial and muscle cells were determined by point counting method. Cardiac hypertrophy and except of 3-week-old rats also BP increase and coronary wall hypertrophy was found in all ontogenic periods in SHR compared to Wistar rats. Arterial wall hypertrophy was evoked by increase of CSA of medial extracellular matrix and smooth muscle cells. In 52-week-old SHR, CSA of muscle cells did not differ from that in 17-week-old SHR but the CSA of intimal and medial extracellular matrix significantly increased. The CSA of endothelial cells and CSA of intimal extracellular matrix were increased only in 52-week-old SHR. The independency between BP and trophicity of individual components of the coronary wall during ontogeny of SHR was documented.