Vascular Lesion Formation Research Articles

Significance of Nitric Oxide Derived from the Nitric Oxide Synthases System in Cardiovascular Inter-Organ Cross-Talk.

Inter-organ cross-talk contributes to the pathogenesis of various disorders, and drug development based on inter-organ cross-talk is attracting attention. The roles of nitric oxide (NO) derived from the NO synthases system (NOSs) in inter-organ cross-talk remain unclear. We have investigated this issue by using our mice deficient in all three NOSs (triple n/i/eNOSs-/- mice). We reported that 2/3 nephrectomized triple n/i/eNOSs-/- mice die suddenly because of early onset of myocardial infarction, suggesting the protective role of NO derived from NOSs in the cross-talk between the kidney and the heart. We studied the role of NO derived from NOSs expressed in the bone marrow in vascular lesion formation. Constrictive arterial remodeling and neointimal formation following unilateral carotid artery ligation were prominently aggravated in wild-type mice transplanted with triple n/i/eNOSs-/- bone marrow cells as compared with those with wild-type bone marrow cells, suggesting the protective role of NO derived from NOSs in the cross-talk between the bone marrow and the blood vessel. We further investigated the role of NO derived from NOSs expressed in the bone marrow in pulmonary hypertension. The extent of pulmonary hypertension after chronic hypoxic exposure was markedly exacerbated in wild-type mice underwent triple n/i/eNOSs-/- bone marrow transplantation as compared with those underwent wild-type bone marrow transplantation, suggesting the protective role of NO derived from NOSs in the cross-talk between the bone marrow and the lung. These lines of evidence demonstrate that systemic and myelocytic NOSs could be novel therapeutic targets for myocardial infarction, vascular disease, and pulmonary hypertension. Significance Statement We demonstrated in studies with triple n/i/eNOSs-/- mice that partial nephrectomy accelerates the occurrence of myocardial infarction induced by systemic NOSs deficiency, that myelocytic NOSs deficiency aggravates vascular lesion formation after unilateral carotid artery ligation, and that myelocytic NOSs deficiency exacerbates chronic hypoxia-induced pulmonary hypertension. These results suggest that NO derived from NOSs plays a protective role in cardiovascular inter-organ cross-talk, indicating that systemic and myelocytic NOSs could be important therapeutic targets for myocardial infarction, vascular disease, and pulmonary hypertension.

Novel Hemodynamic, Vascular Lesion, and Cytokine/Chemokine Differences Regarding Sex in a Pulmonary Arterial Hypertension Model.

Sex differences are recognized in pulmonary hypertension. However, the progression of disease with regard to vascular lesion formation and circulating cytokines/chemokines is unknown. To determine whether vascular lesion formation, changes in hemodynamics, and alterations in circulating chemokines/cytokines differ between males and females, we used a progressive model of pulmonary arterial hypertension (PAH), Sugen/hypoxia, and analyzed cohorts of male and female rats at time points suggested to indicate worsening disease. Our analysis included echocardiography for hemodynamics, morphometry, immunofluoresecence, and chemokine/cytokine analysis of plasma at each time point in both sexes. We found that male rats had significantly increased Fulton index, compared with those for females at each time point, as well as increased medial artery thickening at 8 weeks of PAH. Furthermore, females exhibited fewer obliterative vascular lesions than males at our latest time point. Our data also show increased IL-4, granulocyte-macrophage colony-stimulating factor, IL-10, and macrophage interacting protein-1α that were not observed in females, whereas females were observed to have increased RANTES (whose name derives from Regulated upon Activation, Normal T Cell Expressed and Presumably Secreted) and CXCL-10 that were not found in males. Males also have increased infiltrating macrophages in vascular lesions, compared with females. We found that development of progressive PAH in hemodynamics, morphology, and chemokine/cytokine circulation differs significantly between males and females. These data suggest a macrophage-driven pathology in males, whereas there may be T cell protection from vascular damage in females with PAH.

Abstract 2148: Lung Capillary Endothelium To Arterial Endothelium Transition In Pulmonary Arterial Hypertension

Introduction: Pulmonary arterial hypertension (PAH) is characterized by a progressive increase of pulmonary vascular resistance and obliterative pulmonary vascular remodeling that result in right heart hypertrophy, failure, and premature death. The underlying mechanisms of loss of distal capillary endothelial cells (ECs) and obliterative vascular lesion formation remain unclear. Method and Results: Our recent single-cell RNA sequencing, spatial transcriptomics analysis, RNASCOPE, and immunostaining analysis showed that arterial ECs accumulation and loss of capillary ECs were evident in human PAH patients and pulmonary hypertension (PH) rodents. Pseudotime trajectory analysis of the single-cell RNA sequencing data suggests that lung capillary ECs transit to arterial ECs during the development of PH. Our study also identified CXCL12 as the marker for distal arterial ECs in PH. General capillary EC lineage tracing approach using Plvap-DreERT2; Tdtomato mice demonstrated that general capillary ECs gave rise to arterial ECs during PH development. Genetic deletion of HIF-2a or Notch4 neutralized antibodies normalized the arterial programming in PH. Conclusion: In conclusion, our study demonstrates that general capillary endothelium transits to arterial endothelium through the HIF-2a-Notch4 pathway during the development of PAH. Thus, targeting arterial EC transition might be a novel approach for treating PAH patients. Acknowledgment: This work was supported in part by NIH grants R00HL138278, R01HL158596, R01HL62794, R01HL169509, R01HL170096, AHA Career Development Award 20CDA35310084, The Cardiovascular Research and Education Foundation, Arizona Biomedical Research Centre funding (RFGA2022-01-06), and University of Arizona institution funding to Z.D.

Lung capillary endothelium to arterial endothelium transition in pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is characterized by a progressive increase of pulmonary vascular resistance and obliterative pulmonary vascular remodeling that result in right heart hypertrophy, failure, and premature death. The underlying mechanisms of loss of distal capillary endothelial cells (ECs) and obliterative vascular lesion formation remain unclear. Our recent single-cell RNA sequencing, spatial transcriptomics analysis, RNASCOPE, and immunostaining analysis showed that arterial ECs accumulation and loss of capillary ECs were evident in human PAH patients and pulmonary hypertension (PH) rodents. Pseudotime trajectory analysis of the single-cell RNA sequencing data suggest that lung capillary ECs transit to arterial ECs during the development of PH. Our study also identified CXCL12 as the marker for arterial ECs in PH. General capillary EC lineage tracing approach using Plvap-DreERT2;Tdtomato mice demonstrated that general capillary ECs gave rise to arterial ECs during PH development. Genetic deletion of HIF-2a or pharmacological inhibition of Notch4 normalized the arterial programming in PH. In conclusion, our study demonstrates that general capillary endothelium transits to arterial endothelium through the HIF-2a-Notch4 pathway during the development of PAH. Thus, targeting arterial EC transition might be a novel approach for treating PAH patients. This work was supported in part by NIH grant R00HL138278, R01HL158596, R01HL62794, R01HL169509, R01HL170096, AHA Career Development Award 20CDA35310084, The Cardiovascular Research and Education Foundation, Arizona Biomedical Research Centre funding (RFGA2022-01-06), and University of Arizona institution funding to Z.D. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Protective role of the nitric oxide synthases system in inter-organ communication

The role of the nitric oxide synthases (NOSs) system in inter-organ communication is not fully understood. We addressed this point in our mice lacking all three NOS isoforms (triple n/i/eNOSs-/- mice). We previously reported that the triple n/i/eNOSs-/- mice spontaneously develop myocardial infarction. However, it takes a long time (approximately 1 year) to develop myocardial infarction. We then revealed that 2/3-nephrectomized triple n/i/eNOSs-/- mice suddenly die due to early onset of myocardial infarction, succeeding in developing an experimentally useful model of myocardial infarction. These results suggest the protective role of NOSs in reno-cardiac communication. We next studied the role of NOSs in bone marrow cells (BM) in vascular lesion formation. Constrictive vascular remodeling and neointimal formation at 14 days after carotid artery ligation were markedly accelerated in wild-type (WT) mice transplanted with triple n/i/eNOSs-/- BM as compared with those with WT BM. These results suggest the protective role of NOSs in BM-vascular communication. We then investigated the role of NOSs in BM in pulmonary hypertension (PH). The extents of PH at 3 weeks after hypoxic exposure were markedly exacerbated in WT mice transplanted with triple n/i/eNOSs-/- BM as compared with those with WT BM, suggesting the protective role of NOSs in BM-lung communication. These lines of evidence indicate that systemic and BM NOSs may be a novel therapeutic target in MI, vascular disease, and PH.

A Redox Modulatory SOD Mimetic Nanozyme Prevents the Formation of Cytotoxic Peroxynitrite and Improves Nitric Oxide Bioavailability in Human Endothelial Cells.

The endothelium-derived signalling molecule nitric oxide (NO) in addition to controlling multifarious servo-regulatory functions, suppresses key processes in vascular lesion formation and prevents atherogenesis and other vascular abnormalities. The conversion of NO into cytotoxic and powerful oxidant peroxynitrite (ONOO- ) in a superoxide (O2 .- )-rich environment has emerged as a major reason for reduced NO levels in vascular walls, leading to endothelial dysfunction and cardiovascular complications. So, designing superoxide dismutase (SOD) mimetics that can selectively catalyze the dismutation of O2 .- in the presence of NO, considering their rapid reaction is challenging and is of therapeutic relevance. Herein, the authors report that SOD mimetic cerium vanadate (CeVO4 ) nanozymes effectively regulate the bioavailability of both NO and O2 .- , the two vital constitutive molecules of vascular endothelium, even in the absence of cellular SOD enzyme. The nanozymes optimally modulate the O2 .- level in endothelial cells under oxidative stress conditions and improve endogenously generated NO levels by preventing the formation of ONOO- . Furthermore, nanoparticles exhibit size- and morphology-dependent uptake into the cells and internalize via the clathrin-mediated endocytosis pathway. Intravenous administration of CeVO4 nanoparticles in mice caused no definite organ toxicity and unaltered haematological and biochemical parameters, indicating their biosafety and potential use in biological applications.

Immunological mechanisms of arterial damage in pediatric patients with primary hypertension.

Primary hypertension is a disease that is being diagnosed with increasing frequency in pediatric patients, and many of them are found to have hypertension-mediated organ damage (HMOD), including arterial damage. The pathophysiology of primary hypertension and the formation of HMOD is multifactorial. One mechanism studied in recent years is the subclinical inflammation accompanying the elevation of blood pressure. Experimental studies, studies in adults and children, revealed the involvement of immune mechanisms in the formation of vascular lesions in the course of primary hypertension. The paper summarizes the current knowledge on this subject and points to possible therapeutic targets. Particular emphasis is placed on data from pediatric patients with primary hypertension, as a relation between arterial damage (early vascular aging) and immune system activation had already been found in children. The correct identification of immunological mechanisms may not only broaden our understanding of primary hypertension as a disease but also, more importantly, lead to the most effective methods of its treatment.

The association between white matter hyperintensities and amyloid and tau deposition

White matter hyperintensities (WMHs) frequently occur in Alzheimer’s Disease (AD) and have a contribution from ischemia, though their relationship with β-amyloid and cardiovascular risk factors (CVRFs) is not completely understood. We used AT classification to categorize individuals based on their β-amyloid and tau pathologies, then assessed the effects of β-amyloid and tau on WMH volume and number. We then determined regions in which β-amyloid and WMH accumulation were related. Last, we analyzed the effects of various CVRFs on WMHs. As secondary analyses, we observed effects of age and sex differences, atrophy, cognitive scores, and APOE genotype. PET, MRI, FLAIR, demographic, and cardiovascular health data was collected from the Alzheimer’s Disease Neuroimaging Initiative (ADNI-3) (N = 287, 48 % male). Participants were categorized as A + and T + if their Florbetapir SUVR and Flortaucipir SUVR were above 0.79 and 1.25, respectively. WMHs were mapped on MRI using a deep convolutional neural network (Sepehrband et al., 2020). CVRF scores were based on history of hypertension, systolic and diastolic blood pressure, pulse rate, respiration rate, BMI, and a cumulative score with 6 being the maximum score. Regression models and Pearson correlations were used to test associations and correlations between variables, respectively, with age, sex, years of education, and scanner manufacturer as covariates of no interest. WMH volume percent was significantly associated with global β-amyloid (r = 0.28, p < 0.001), but not tau (r = 0.05, p = 0.25). WMH volume percent was higher in individuals with either A + or T + pathology compared to controls, particularly within in the A+/T + group (p = 0.007, Cohen’s d = 0.4, t = -2.5). Individual CVRFs nor cumulative CVRF scores were associated with increased WMH volume. Finally, the regions where β-amyloid and WMH count were most positively associated were the middle temporal region in the right hemisphere (r = 0.18, p = 0.002) and the fusiform region in the left hemisphere (r = 0.017, p = 0.005). β-amyloid and WMH have a clear association, though the mechanism facilitating this association is still not fully understood. The associations found between β-amyloid and WMH burden emphasizes the relationship between β-amyloid and vascular lesion formation while factors like CVRFs, age, and sex affect AD development through various mechanisms. These findings highlight potential causes and mechanisms of AD as targets for future preventions and treatments. Going forward, a larger emphasis may be placed on β-amyloid’s vascular effects and the implications of impaired brain clearance in AD.

Malondialdehyde as an Important Key Factor of Molecular Mechanisms of Vascular Wall Damage under Heart Diseases Development.

This mini review is devoted to a specific issue: the role of malondialdehyde (MDA)-a secondary product of free radical lipid peroxidation-in the molecular mechanisms of the formation of primary atherosclerotic vascular wall lesions. The principal difference between this review and the available literature is that it discusses in detail the important role in atherogenesis not of "oxidized" LDL (i.e., LDL particles containing lipohydroperoxides), but of LDL particles chemically modified by the natural low-molecular weight dicarbonyl MDA. To confirm this, we consider the data obtained by us earlier, indicating that "atherogenic" are not LDL oxidized as a result of free radical lipoperoxidation and containing lipohydroperoxy derivatives of phospholipids in the outer layer of particles, but LDL whose apoprotein B-100 has been modified due to the chemical reaction of terminal lysine residue amino groups of the apoB-100 with the aldehyde groups of the MDA (Maillard reaction). In addition, we present our original data proving that MDA injures endothelial glycocalyx that suppress the ability of the endothelium to control arterial tone according to changes in wall shear stress. In summary, this mini review for the first time exhaustively discloses the key role of MDA in atherogenesis.

An update on preclinical models of hereditary haemorrhagic telangiectasia: Insights into disease mechanisms.

Endoglin (ENG) is expressed on the surface of endothelial cells (ECs) where it efficiently binds circulating BMP9 and BMP10 ligands to initiate activin A receptor like type 1 (ALK1) protein signalling to protect the vascular architecture. Patients heterozygous for ENG or ALK1 mutations develop the vascular disorder known as hereditary haemorrhagic telangiectasia (HHT). Many patients with this disorder suffer from anaemia, and are also at increased risk of stroke and high output heart failure. Recent work using animal models of HHT has revealed new insights into cellular and molecular mechanisms causing this disease. Loss of the ENG (HHT1) or ALK1 (HHT2) gene in ECs leads to aberrant arteriovenous connections or malformations (AVMs) in developing blood vessels. Similar phenotypes develop following combined EC specific loss of SMAD1 and 5, or EC loss of SMAD4. Taken together these data point to the essential role of the BMP9/10-ENG-ALK1-SMAD1/5-SMAD4 pathway in protecting the vasculature from AVMs. Altered directional migration of ECs in response to shear stress and increased EC proliferation are now recognised as critical factors driving AVM formation. Disruption of the ENG/ALK1 signalling pathway also affects EC responses to vascular endothelial growth factor (VEGF) and crosstalk between ECs and vascular smooth muscle cells. It is striking that the vascular lesions in HHT are both localised and tissue specific. Increasing evidence points to the importance of a second genetic hit to generate biallelic mutations, and the sporadic nature of such somatic mutations would explain the localised formation of vascular lesions. In addition, different pro-angiogenic drivers of AVM formation are likely to be at play during the patient’s life course. For example, inflammation is a key driver of vessel remodelling in postnatal life, and may turn out to be an important driver of HHT disease. The current wealth of preclinical models of HHT has led to increased understanding of AVM development and revealed new therapeutic approaches to treat AVMs, and form the topic of this review.

Surgical Outcome of Potassium-Titanyl-Phosphate (KTP) Laser Photocoagulation for Vocal Fold Vascular Lesions

IntroductionLaser technology is used in microscopic direct laryngeal surgery for a variety of indications. Lasers are categorized broadly as photoangiolytic or cutting/ablating lasers, based on the chromophores that absorb their energy. Photoangiolytic lasers such as the 532 nm Potassium-Titanyl-Phosphate (KTP) laser are absorbed selectively by the chromophore hemoglobin, facilitating controlled intravascular coagulation, with preservation of the overlying epithelium and adjacent tissue. Efficacy of the KTP laser has been demonstrated for incision, coagulation, and ablation in vocal fold (VF) surgery. ObjectiveThe purpose of the present study was to examine surgical outcomes following KTP laser photocoagulation for the management of VF vascular lesions. MethodsAdult patients with sufficient data who had undergone KTP laser photocoagulation in the operating room for the treatment of VF vascular lesions were included in this retrospective study. Strobovideolaryngoscopy (SVL) video footage from all preoperative visits and all available postoperative visits was compiled and de-identified. Patients were followed up at days 1-7, 8-14, 30-60, and greater than 60 days after surgery. Three blinded physician evaluators reviewed and evaluated the SVL footage independently for postoperative outcome parameters. Postoperative SVL video footage was evaluated on a 5-point scale for surgical success (surgical objective score; 1 = failure and 5 = complete success). The average surgical objective score was 4.36, 4.04, 4.25, and 4.46 (out of 5) at postoperative visits 1-4, respectively. ResultsThere were 60 cases (19 male and 41 female) included in the retrospective cohort. The average age was 42.42 ± 15.51 (range = 18-74). Fifty-one-point six seven percent of subjects were professional voice users (singers, teachers, public speakers, and others). All subjects were diagnosed preoperatively with VF vascular malformations and had undergone pulsed KTP laser photocoagulation. There were 40 bilateral cases and 20 unilateral cases, for a total of 100 VFs included in the study. Vascular malformation recurrence was identified in 3.00% and 10.00% of subjects at the third and fourth postoperative visits, retrospectively. The formation of new vascular malformations was identified in 0.00%, 1.00%, 6.00%, and 7.00% of subjects at postoperative visits 1-4, respectively. ConclusionKTP laser photocoagulation is highly effective for the management of vocal fold vascular lesions. Presence of hemorrhage or edema does not affect the long-term surgical outcome. In relatively few cases, vascular lesion recurrence or formation of new vascular lesions may occur.

High-phosphorus diets reduce aortic lesions and cardiomyocyte size and modify lipid metabolism in Ldl receptor knockout mice

The consumption of phosphorus in Western populations largely exceeds the recommended intake, while vitamin D supply is often insufficient. Both situations are linked to an increased cardiovascular risk. A 17-week two-factorial study with Ldl receptor-/- mice was conducted to investigate the cardiovascular impact of dietary phosphorus [adequate (0.3%; P0.3) vs. high (1.5%; P1.5)] in combination with a low (50 IU/kg; D50) or adequate vitamin D diet (1000 IU/kg; D1000). The data demonstrate that mice fed the P1.5 vs. P0.3 diets developed smaller vascular lesions (p = 0.013) and cardiac hypotrophy (p = 0.011), which were accompanied by diminished IGF1 and insulin signalling activity in their hearts. Vitamin D showed no independent effect on atherogenesis and heart morphology. Feeding P1.5 vs. P0.3 diets resulted in markedly reduced serum triacylglycerols (p < 0.0001) and cholesterol (p < 0.0001), higher faecal lipid excretion (p < 0.0001) and a reduced mRNA abundance of hepatic sterol exporters and lipoprotein receptors. Minor hypocholesterolaemic and hypotriglyceridaemic effects were also found in mice fed the D1000 vs. D50 diets (p = 0.048, p = 0.026). To conclude, a high phosphorus intake strongly affected the formation of vascular lesions, cardiac morphology, and lipid metabolism, although these changes are not indicative of an increased cardiovascular risk.

Endothelial cells and endothelial progenitor cells in the pathogenesis of systemic sclerosis.

Systemic sclerosis (SSc) is a connective tissue disease characterized by excessive fibrosis, microvasculopathy, and autoimmunity. Endothelial cell (EC) injury and subsequent endothelial cell dysfunction is believed to be an initial event that eventually leads to a vicious pathogenic cycle. This process is further enhanced by defective angiogenesis and vasculogenesis, as the vascular repair machinery does not work properly. Endothelial progenitor cells (EPCs) are functionally and quantitatively insufficient to recover the endothelium in SSc patients. The dysfunctional ECs and EPCs not only trigger the formation of typical vascular lesions, such as progressive intimal fibrosis in small arteries and the loss of capillaries, but also promote a series of inflammatory and profibrotic processes, such as endothelial-mesenchymal transition and recruitment and accumulation of monocytic EPCs with profibrotic properties. These processes together contribute to the accumulation of extracellular matrix in the affected tissue. This review features current insights into the roles of ECs and EPCs in the pathogenesis of SSc.

Genetics of vascular anomalies

Vascular anomalies are developmental defects of the vasculature and encompass a variety of disorders. The identification of genes mutated in the different malformations provides insight into the etiopathogenic mechanisms and the specific roles the associated proteins play in vascular development and maintenance. A few familial forms of vascular anomalies exist, but most cases occur sporadically. It is becoming evident that somatic mosaicism plays a major role in the formation of vascular lesions. The use of Next Generating Sequencing for high throughput and “deep” screening of both blood and lesional DNA and RNA has been instrumental in detecting such low frequency somatic changes. The number of novel causative mutations identified for many vascular anomalies has soared within a 10-year period. The discovery of such genes aided in unraveling a holistic overview of the pathogenic mechanisms, by which in vitro and in vivo models could be generated, and opening the doors to development of more effective treatments that do not address just symptoms. Moreover, as many mutations and the implicated signaling pathways are shared with cancers, current oncological therapies could potentially be repurposed for the treatment of vascular anomalies.

Loss of the transcription factor RBPJ induces disease-promoting properties in brain pericytes

Sufficient vascular supply is indispensable for brain development and function, whereas dysfunctional blood vessels are associated with human diseases such as vascular malformations, stroke or neurodegeneration. Pericytes are capillary-associated mesenchymal cells that limit vascular permeability and protect the brain by preserving blood-brain barrier integrity. Loss of pericytes has been linked to neurodegenerative changes in genetically modified mice. Here, we report that postnatal inactivation of the Rbpj gene, encoding the transcription factor RBPJ, leads to alteration of cell identity markers in brain pericytes, increases local TGFβ signalling, and triggers profound changes in endothelial behaviour. These changes, which are not mimicked by pericyte ablation, imperil vascular stability and induce the acquisition of pathological landmarks associated with cerebral cavernous malformations. In adult mice, loss of Rbpj results in bigger stroke lesions upon ischemic insult. We propose that brain pericytes can acquire deleterious properties that actively enhance vascular lesion formation and promote pathogenic processes.

Roles of Perivascular Adipose Tissue in the Pathogenesis of Atherosclerosis.

Traditionally, it is believed that white adipose tissues serve as energy storage, heat insulation, and mechanical cushion, whereas non-shivering thermogenesis occurs in brown adipose tissue. Recent evidence revealed that adipose tissue secretes many types of cytokines, called as adipocytokines, which modulate glucose metabolism, lipid profile, appetite, fibrinolysis, blood pressure, and inflammation. Most of the arteries are surrounded by perivascular adipose tissue (PVAT). PVAT has been thought to be simply a structurally supportive tissue for vasculature. However, recent studies showed that PVAT influences vasodilation and vasocontraction, suggesting that PVAT regulates vascular tone and diameter. Adipocytokines secreted from PVAT appear to have direct access to the adjacent arterial wall by diffusion or via vasa vasorum. In fact, PVAT around atherosclerotic lesions and mechanically-injured arteries displayed inflammatory cytokine profiles, suggesting that PVAT functions to promote vascular lesion formation. Many clinical studies revealed that increased accumulation of epicardial adipose tissue (EAT), which surrounds coronary arteries, is associated with coronary artery disease. In this review article, we will summarize recent findings about potential roles of PVAT in the pathogenesis of atherosclerosis, particularly focusing on a series of basic and clinical studies from our laboratory.

Development and Multiparametric Evaluation of Experimental Atherosclerosis in Rabbits.

Several animal models have been developed to study atherosclerosis. Here we present a rabbit atherosclerosis model generated by surgical denudation of the aortic endothelium in combination with a high-fat and cholesterol-enriched diet. This model is characterized by the formation of vascular lesions that exhibit several hallmarks of human atherosclerosis. Due to the rabbit's relative large size, as compared to rodents, this model is suited for the imaging-guided evaluation of novel therapeutic strategies using clinical scanners. In this chapter, we present an extensive outline of the procedures to induce aortic atherosclerotic lesions in rabbits as well as methods to evaluate the disease, including noninvasive in vivo multiparametric imaging and histopathology.

Perivascular mast cells promote neointimal elastin deposition and suppress chronic vein graft restenosis in hyperlipidaemic mice

Aims: Mast cells are versatile innate immune cells and are reported to promote vascular inflammation and neointimal lesion formation, thereby contributing to the development of vascular stenosis and atherosclerosis. However, it is not clear whether mast cells also regulate vascular matrix remodelling in established neointima. This study addressed the hypothesis that perivascular mast cells regulate neointimal matrix remodelling using a mouse vein graft model. Methods: The impact of mast cells on neointimal remodelling was investigated using mast cell-deficient animals in both normolipidaemic (KitW-sh/W-sh) and hyperlipidaemic (apoE-/-KitW-sh/W-sh) conditions. The effect of perivascular mast cells on vascular matrix remodelling, including collagen and elastin deposition, was investigated using a local mast cell reconstitution method that selectively repopulated mast cells around the carotid artery (where the vein graft was inserted) in KitW-sh/W-sh mice. Results: In normolipidaemic vein grafts (KitW-sh/W-sh vs. the wild type control C57BL/6J), collagen synthesis was not affected by mast cell deficiency at 4 weeks. In contrast, neointimal elastin was reduced by 6.5-fold in mast cell-deficient KitW-sh/W-sh mice, which was prevented by perivascular mast cell reconstitution. Mast cell deficiency induced a similar reduction in neointimal elastin in hyperlipidaemic mice (apoE-/-KitW-sh/W-sh vs. apoE-/-), with a significant increase in cell proliferation and neointimal area at 4 week. Conclusion: Mast cells appear to promote elastin deposition in vein grafts and this may lead to further suppression of cell proliferation and neointimal thickening under hyperlipidaemic conditions.

Docosahexaenoic Acid Inhibits Vascular Smooth Muscle Cell Proliferation Induced by Glucose Variability.

Background:Vascular Smooth Muscle cells (VSMC) enact crucial roles in early vasculogenesis and sustenance of vascular integrity. However, aberrant proliferation of VSMC followed by migration into the blood vessel wall leads to the formation of vascular lesions accounting for the degeneration and remodelling of vascular basement membrane. In diabetes, hyperglycaemia accelerates VSMC proliferation and contributes to the initiation and progression of atherosclerotic lesions. Recently, acute glucose fluctuations have been implicated in the abnormal VSMC proliferation and complications of diabetic atherosclerosis. Docosahexaenoic acid (DHA), a ω-3 polyunsaturated fatty acid (PUFA) has been shown to inhibit proliferation of several cell types implicating several different mechanisms. In the present study, we have investigated the effects of DHA on VSMC proliferation induced by stable and intermittent high glucose levels.Method:Confluent cultures of rat aortic VSMCs were treated with DHA for 24 hrs and then exposed to stable high glucose (25 mmol/L, SHG) or intermittent high glucose (5 mmol/L and 25 mmol/L alternating every 12 hrs, IHG) for 72 hrs. Cell proliferation was examined by the MTT viability assay, while apoptosis process was evaluated by the Hoechst staining, flow cytometry and caspase-3 activity assays.Results:Our data demonstrated that the hyper proliferation induced by stable and intermittent high glucose levels was significantly inhibited by the DHA pre-treatment. DHA significantly increased caspase-3 activity, resulting in enhanced DNA fragmentation and apoptosis.Conclusion:Our results suggest that DHA reduced the high glucose-induced proliferation of VSMC and induced cell apoptosis.

Artificial Zinc-Finger Transcription Factor of A20 Suppresses Restenosis in Sprague Dawley Rats after Carotid Injury via the PPARα Pathway.

The inhibition of inflammation and vascular smooth muscle cell (VSMC) proliferation is an ideal strategy to suppress intimal hyperplasia after percutaneous transluminal angioplasty (PTA). Evidence has indicated that overexpression of A20 suppresses neointima formation, but its low transfection efficiency limits its application. Hence, we upregulated A20 expression via transfection of rAd.ATF (recombinant adenovirus vector of artificial transcription factor) and rAd.A20 in rat carotid arteries after balloon dilatation (in vivo) and isolated VSMCs (in vitro). In vivo, we found that after rAd.ATF and rAd.A20 transfection, A20 expression was markedly increased, whereas proliferating cell nuclear antigen (PCNA) and nuclear factor κB p65 (NF-κBp65) protein levels were significantly decreased, and intimal hyperplasia and secretion of proinflammatory factors were significantly reduced when compared with empty vector and saline control groups. Most importantly, the rAd.ATF-treated group showed more significant inhibition on intimal hyperplasia and expression of PCNA than the rAd.A20-treated group. In vitro, compared with the control group, transfection of rAd.ATF and rAd.A20 significantly increased A20 expression, which upregulated the proliferator-activated receptor (PPAR) level for both mRNA and protein, and reduced migration and proliferation of VSMCs and lipopolysaccharide (LPS)-induced inflammation. Furthermore, the PPARα agonist GW6471 could partially restore the effect of A20 on VSMCs. Our findings indicate that the ATF of A20 inhibits neointimal hyperplasia and, therefore, constitutes a novel potential alternative to prevent restenosis.