Local Delivery of Pravastatin Inhibits Intimal Formation in a Mouse Vein Graft Model

Background

Pravastatin can reduce atherosclerotic progression in patients after coronary artery bypass graft. However, it is unknown whether pravastatin has a direct effect on intimal hyperplasia of grafted vessels in vivo or what the underlying mechanisms may be. In this study, a murine vein graft model was applied to deal with these issues.

Methods

Vein grafting was performed between C57BL/6J mice. Immediately after operation, pravastatin (30 μM) or phosphate-buffered saline in 50 μL 20% pluronic F-127 gel was delivered to the adventitia of grafted vessels.

Results

Compared with the vehicle, pravastatin significantly reduced intimal hyperplasia 4 weeks after the surgical procedure. Immunohistochemical studies revealed that vascular smooth muscle cells (VSMCs) are a major component of the neointima. The percentage of cells positive for proliferating cell nuclear antigen and Mac-3-positive immunostaining intensity within the intima of vein grafts was significantly lower in the pravastatin-treated group than in the control group. We separated VSMCs from mouse inferior vena cava and collected peritoneal macrophage from mice injected intraperitoneally with 4% thioglycollate. Pravastatin significantly decreased VSMC proliferation and platelet-derived growth factor–induced VSMC migration and, in a dose-dependent manner, inhibited macrophage migration induced by monocyte chemotactic protein-1.

Conclusions

Local delivery of pravastatin at the time of vein-graft surgery directly suppresses subsequent neointimal formation of grafted vessels in a vein graft model of normocholesterolemic mice. These beneficial effects are associated with inhibitory actions on VSMC and macrophage functions.     

Nalp3 inflammasome is activated and required for vascular smooth muscle cell calcification

Background

The calcification of blood vessels correlates with increased morbidity and mortality in patients with atherosclerosis, diabetes, and end-stage kidney disease. Increased inflammasome activation has been shown to play an important role in the pathogenesis of atherosclerosis. However, the contribution of inflammasome activation on the development of vascular calcification has not been investigated.

Methods

β-Glycerophosphate (β-GP) was used as a procedure to induce extensive artery calcification in primary vascular smooth muscle cells (VSMCs). Analysis of the levels of Nalp3 inflammasome complex was performed by quantitative real-time PCR and western blotting. The effect of Nalp3 deficiency on VSMC calcification was examined after transfecting Nalp3 siRNA into cultured VSMCs.

Results

We demonstrated for the first time that the mRNA levels of Nalp3 inflammasome complex including Nalp3, ASC and caspase1 were upregulated in calcifying VSMCs, resulting in increased IL-1β secretion. Inhibition of inflammasome activation by Nalp3 RNA interference reduced IL-1β secretion and inhibited VSMC calcification. Further analysis of clinical popliteal artery specimens showed an upregulation of inflammasome complex mRNA levels (4/5) and caspase1 activity (5/5) compared with their non-calcified adjacent tissues, indicating that Nalp3 inflammasome was tightly correlated with arterial calcification disease.

Conclusion

Our findings indicate that activation of the Nalp3-mediated inflammatory response pathway is an important venue associated with host response and pathogenesis of VSMC calcification.     

MicroRNA-133a regulates insulin-like growth factor-1 receptor expression and vascular smooth muscle cell proliferation in murine atherosclerosis

Objective

MicroRNA-133a (miR-133a) and insulin-like growth factor-1 (IGF-1) are two different molecules known to regulate cardiovascular cell proliferation. This study tested whether miR-133a affects expression of IGF-1 receptor (IGF-1R) and proliferation of IGF-1-stimulated vascular smooth muscle cells (VSMC) in a murine model of atherosclerosis.

Methods and results

Expression of IGF-1R was analyzed by immuno-fluorescence and immuno-blotting, and miR-133a by qRT-PCR in the aortas of wild-type C57BL/6J (WT) and apolipoprotein-E deficient (ApoE^−/−) mice. Compared to those in WT aortas, the IGF-1R and miR-133a levels were lower in ApoE^−/− aortas. ApoE^−/− VSMC grew slower than WT cells in the cultures with IGF-1-containing medium. MiR-133a-specific inhibitor decreased miR-133a, IGF-1R expression, IGF-1-stimulated VSMC growth in lipoprotein deficient media. By contrast, miR-133a precursor increased IGF-1R levels and promoted IGF-1-induced VSMC proliferation. In the luciferase-IGF-1R 3′UTR reporter system, the reporter luciferase activity was not inhibited in VSMC with miR-133a overexpression. IGF-1R mRNA half-life in ApoE^−/− VSMC was shorter than that in WT VSMC. MiR-133a inhibitor reduced but precursor increased the mRNA half-life, although the effects appeared less striking in ApoE^−/− VSMC than in WT cells.

Conclusion

MiR-133a serves as a stimulatory factor for IGF-1R expression through prolonging IGF-1R mRNA half-life. In atherosclerosis induced by ApoE deficiency, reduced miR-133a expression is associated with lower IGF-1R levels and suppressive VSMC growth. Administration of miR-133a precursor may potentiate IGF-1-stimulated VSMC survival and growth.     

Influence of apolipoprotein E,age and aortic site on calcium phosphate induced abdominal aortic aneurysm in mice

Objective

To assess relevant features of abdominal aortic aneurysms (AAA) induced by calcium phosphate within a mouse model. Specifically we investigated: (1) whether apolipoprotein E deficiency and older age promoted AAA formation, and (2) whether the local application of calcium phosphate affected the size of distant aortic segments.

Methods

AAA was induced by application of calcium phosphate to the infra-renal aortas of 3 and 7 month old male mice. AAA induction was assessed by calculating expansion of the infra-renal aortic diameter over 1–4 weeks. Aortic samples were assessed to quantify calcification, macrophages infiltration, elastic lamellar degradation and apoptosis. Blood pressure was measured by the tail cuff method, and plasma concentrations of total cholesterol, low density lipoprotein and very low density lipoprotein cholesterol, and pro-inflammatory cytokines were measured using commercially available kits. The maximum diameters of the aortic arch, thoracic and supra-renal aorta at sacrifice were measured by morphometry and the mean maximal diameter of these three aortic segments was calculated.

Results

The median expansion of the infra-renal aorta 2 weeks after AAA induction was significantly greater in apolipoprotein E deficient (ApoE^−/−) mice than in age- and gender-matched wild type controls [275.8% (IQR 193.8%–348.5%) versus 94.7% (IQR 47.8%–163.4%), P = 0.02]. The greater aortic expansion in ApoE^−/− mice was associated with aortic calcification, macrophage infiltration, elastic lamellar degradation and apoptosis of cells in the media and adventitia. The plasma low density lipoprotein/very low density lipoprotein cholesterol concentrations 2 weeks after AAA induction were positively correlated with the expansion of the infra-renal aorta induced by calcium phosphate. The median expansion of the infra-renal aorta 2 weeks after AAA induction was similar in 3 and 7 month old wild type mice. The local administration of calcium phosphate was associated with an increase in the mean maximal diameter of distant aortic segments, but not associated with changes in the concentrations of pro-inflammatory markers in either the plasma or the spleen.

Conclusion

This study suggests that apolipoprotein E deficiency, but not age, predisposes to AAA induced within the calcium phosphate model. Increased AAA expansion in ApoE^−/− mice was associated with calcification, macrophage infiltration, elastic lamellar degradation, and cell apoptosis. Local application of calcium phosphate also promoted dilation of distant aortic segments.     

The LDLR deficient mouse as a model for aortic calcification and quantification by micro-computed tomography

Objective

Patients with familial hypercholesterolemia (FH) due mutations in the low-density lipoprotein receptor (LDLR) suffer premature aortic calcification, an effect that is age- and gene dosage-dependent and cholesterol level independent later in life. To better understand this process, we examined a murine model.

Methods

We compared chow fed Ldlr^−/− mice to controls at 6, 12 and 18 months and on a Western diet (WD) at 6 months. Additionally, we compared controls to Ldlr^−/− mice and transgenic mice Tg(Pcsk9) overexpressing PCSK9, which promotes LDLR degradation. Aortas were perfused-fixed, embedded in paraffin, and sections were stained with alizarin red. Micro-computerized tomography (micro-CT) was used to quantify vascular calcification.

Results

Ldlr^−/− mice develop calcification in the ascending, transverse aorta and neck vessels with a distribution similar to that of human. Calcification was most prominent in 18-month-old Ldlr^−/− mice fed a chow diet and in 6-month-old Ldlr^−/− mice fed a WD. Interestingly, Tg(Pcsk9) mice fed a WD develop aortic calcifications as well. Histology confirmed that the calcification were predominantly sub-intimal. Marked expression of LRP5 and WNT was observed in the Ldlr^−/− and Tg(Pcsk9) models, but not in age-matched controls.

Conclusions

The two mouse models develop aortic calcification in an age- and diet-dependent manner. Abnormal regulation of the LRP5/Wnt pathway may play a role in the calcification process. Further analysis of these aortic calcification models using this micro-CT imaging technique may provide a better understanding of the link between FH and arterial calcification.     

Adiponectin attenuates abdominal aortic aneurysm formation in hyperlipidemic mice

Objective

Abdominal aortic aneurysms (AAA) are age-associated, life-threatening inflammatory dilations of the abdominal aorta. Human population studies have shown an association between obesity and AAA formation, but the molecular mechanisms underlying this connection remain largely unexplored. Adiponectin is an anti-inflammatory adipokine that is downregulated in obesity. In this study we evaluated the role of adiponectin in a model of AAA using apolipoprotein E/adiponectin double-knockout (Apoe^−/−Apn^−/−) mice.

Approach and results

Angiotensin II (Ang II)-infusion in male Apoe^−/−Apn^−/− mice led to a higher incidence of AAA and a significant increase of maximal aortic diameter compared with that of Apoe^−/− mice (2.12 ± 0.07 mm vs. 1.67 ± 0.09 mm, respectively at 28 days). Adiponectin deficiency augmented the early infiltration of macrophages and increased the expression of pro-inflammatory factors in the dilated aortic wall. MMP-2 and MMP-9 activation was also augmented in the aorta of Apoe^−/−Apn^−/− mice compared to Apoe^−/− mice. These data suggest that the downregulation of adiponectin could directly contribute to the elevated incidence of AAA observed in obese individuals.

Conclusions

Adiponectin attenuates Ang II-induced vascular inflammation and AAA formation in mice.     

Coronary artery calcification correlates with the presence and severity of valve calcification

Aim

To investigate the prevalence of coronary artery calcification (CAC) in symptomatic individuals with CT evidence for left heart valve calcification, aortic valve (AVC), mitral valve (MAC) or both.

Methods

This is a retrospective study of 282 consecutive patients with calcification in either the aortic valve or mitral annulus. Calcium scoring of the coronary artery, aortic and mitral valve was measured using the Agatston score.

Results

AVC was more prevalent than MAC (64% vs. 2.5%, p < 0.001), with 34% having both. Absence of CAC was noted in 12.7% of the study population. AVC + CAC were observed in 53.5%, MAC and CAC in 2.1%, and combined AVC, MAC and CAC in 31.6%. The median CAC score was higher in individuals with combined AVC + MAC, followed by those with AVC and lowest was in the MAC group. The majority (40%) of individuals with AVC had CAC score > 400, and only in 16% had CAC = 0. The same pattern was more evident in individuals with AVC + MAC, where 70% had CAC score > 400 and only 6% had CAC score of 0. These results were irrespective of gender. There was no correlation between AVC and MAC but there was modest correlation between CAC score and AVC score (r = 0.28, p = 0.0001), MAC (r = 0.36, p = 0.0001) and with combined AVC + MAC (r = 0.5, p = 0.0001). AVC score of 262 had a sensitivity of 78% and specificity of 92% for the prediction of presence of CAC.

Conclusion

The presence and extent of calcification in the aortic valve or/and mitral valves are associated with severe coronary artery calcification.     

HGF/c-Met signalling promotes Notch3 activation and human vascular smooth muscle cell osteogenic differentiation in vitro

Objectives

Vascular calcification is a major clinical problem and elucidating the underlying mechanism is important to improve the prognosis of patients with cardiovascular disease. We aimed to elucidate the role and mechanism of action of Hepatocyte Growth Factor (HGF)/c-Met signalling in vascular calcification and establish whether blocking this pathway could prevent mineralisation of vascular smooth muscle cells (VSMCs) in vitro.

Methods and results

We demonstrate increased HGF secretion and c-Met up-regulation and phosphorylation during VSMC osteogenic differentiation. Adenoviral-mediated over-expression of HGF (AdHGF) in VSMCs accelerated mineralisation, shown by alizarin red staining, and significantly increased ⁴⁵Calcium incorporation (1.96 ± 0.54-fold [P < 0.05]) and alkaline phosphatase (ALP) activity (3.01 ± 0.8-fold [P < 0.05]) compared to controls. AdHGF also significantly elevated mRNA expression of bone-related proteins, Runx2, osteocalcin, BMP2 and osterix in VSMCs. AdHGF-accelerated mineralisation correlated with increased Akt phosphorylation, nuclear translocation of Notch3 intracellular domain (N3IC) and up-regulation of the Notch3 target protein, HES1. In contrast, adenoviral-mediated over-expression of the HGF antagonist, NK4, markedly attenuated VSMC mineralisation, and reduced c-Met phosphorylation, Akt activation and HES1 protein expression compared to AdHGF-treated cells. Furthermore, the Notch inhibitor, DAPT, attenuated N3IC nuclear translocation and AdHGF-induced mineralisation.

Conclusion

We demonstrate HGF induces VSMC osteogenic differentiation via c-Met/Akt/Notch3 signalling, highlighting these pathways as potential targets for intervention of vascular calcification.     

The Mammalian Target of Rapamycin Signalling Pathway Is Involved in Osteoblastic Differentiation of Vascular Smooth Muscle Cells

Background

Vascular calcification is a major risk factor for cardiovascular diseases. Osteoblastic differentiation of vascular smooth muscle cells (VSMCs) is a key step in vascular calcification, but the molecular mechanisms driving the differentiation remain elusive. In this study, the involvement of mammalian target of rapamycin (mTOR) signalling in osteoblastic differentiation of VSMCs is investigated.

Methods

Calcification of VSMCs was induced in vitro using β-glycerophosphate (β-GP). Real-time polymerase chain reaction was used to measure messenger RNA (mRNA) expression, and Western blot was used to detect protein expression. Inhibition of mTOR expression was established by small interfering RNA (siRNA) and mTOR inhibitors.

Results

The model for osteoblastic differentiation of VSMCs was established in vitro by treating mouse VSMCs with 10 mM β-GP for 3-15 days. Overexpression of mTOR was observed in differentiated VSMCs. Downregulation of mTOR by siRNA or rapamycin significantly inhibited osteoblastic differentiation of VSMCs and decreased the expression and phosphorylation of mTOR and P70 ribosomal S6 kinase in a time- and concentration-dependent manner. Furthermore, adiponectin inhibited the mRNA and protein expression of mTOR in β-GP-treated VSMCs in a time- and concentration-dependent manner.

Conclusions

mTOR signalling plays a crucial role in the osteoblastic differentiation of VSMCs. Rapamycin and adiponectin might inhibit vascular calcification through regulation of the mTOR pathway.     

ER stress signalling through eIF2α and CHOP, but not IRE1α, attenuates adipogenesis in mice

Aims/hypothesis

Although obesity is associated with endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) in adipose tissue, it is not known how UPR signalling affects adipogenesis. To test whether signalling through protein kinase RNA-like ER kinase/eukaryotic initiation factor 2 alpha (PERK/eIF2α) or inositol-requiring enzyme 1 alpha/X-box binding protein 1 (IRE1α/XBP1) is required for adipogenesis, we studied the role of UPR signalling in adipocyte differentiation in vitro and in vivo in mice.

Methods

The role of UPR signalling in adipogenesis was investigated using 3T3-L1 cells and primary mouse embryonic fibroblasts (MEFs) by activation or inhibition of PERK-mediated phosphorylation of the eIF2α- and IRE1α-mediated splicing of Xbp1 mRNA. Body weight change, fat mass composition and adipocyte number and size were measured in wild-type and genetically engineered mice fed a control or high-fat diet (HFD).

Results

ER stress repressed adipocyte differentiation in 3T3-L1 cells. Impaired eIF2α phosphorylation enhanced adipocyte differentiation in MEFs, as well as in mice. In contrast, increased eIF2α phosphorylation reduced adipocyte differentiation in 3T3-L1 cells. Forced production of CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP), a downstream target of eIF2α phosphorylation, inhibited adipogenesis in 3T3-L1 cells. Mice with deletion of Chop (also known as Ddit3) (Chop ^?/?) gained more fat mass than wild-type mice on HFD. In addition, Chop deletion in genetically obese Lepr ^db/db mice increased body fat mass without altering adipocyte size. In contrast to the eIF2α–CHOP pathway, activation or deletion of Ire1a (also known as Ern1) did not alter adipocyte differentiation in 3T3-L1 cells.

Conclusions/interpretation

These results demonstrate that eIF2α–CHOP suppresses adipogenesis and limits expansion of fat mass in vivo in mice, rendering this pathway a potential therapeutic target.     

Congenital Absence of Nitric Oxide Synthase 3 Potentiates Cardiac Dysfunction and Reduces Survival in Doxorubicin- and Trastuzumab-Mediated Cardiomyopathy

Background

Doxorubicin (DOX) and trastuzumab (TRZ) are highly effective chemotherapeutic agents in the breast cancer setting, limited by their cardiotoxic side effects. Among the potential mechanisms for this drug-induced cardiomyopathy, increased production of oxidative stress (OS) through a nitric oxide synthase 3 (NOS3)-dependent pathway has gained recent attention. The objective of the study was to determine the role of NOS3 and OS in a clinically relevant female murine model of DOX- and TRZ-induced heart failure.

Methods

A total of 120 female mice (60 wild-type [WT] and 60 NOS3 knockout [NOS3^−/−]) were treated with either 0.9% saline, DOX, TRZ, or DOX with TRZ (DOX+TRZ). Serial echocardiography was performed for a total of 10 days, after which the mice were euthanized for histological and biochemical analyses.

Results

In WT female mice receiving DOX+TRZ, left ventricular ejection fraction (LVEF) decreased from 75 ± 3% at baseline to 46 ± 2% at day 10 (P < 0.05). In the NOS3^−/− group, LVEF decreased from 72 ± 3% at baseline to 35 ± 2% at day 10 (P < 0.05). LVEF was significantly lower in NOS3^−/− female mice receiving DOX+TRZ than WT mice at day 10 (P < 0.05). Compared with WT, NOS3^−/− female mice also demonstrated increased mortality after treatment with DOX+TRZ, corroborating the echocardiographic findings. Histological analysis demonstrated increased myofibrillar degradation and loss of cell integrity in NOS3^−/− female mice treated with DOX+TRZ. There was increased generation of oxidized phosphatidylcholine, a marker of OS, in NOS3^−/− female mice receiving DOX+TRZ compared with control mice.

Conclusions

Congenital absence of NOS3 potentiates the cardiotoxic side effects of DOX+TRZ in an acute female murine model of chemotherapy-induced cardiomyopathy.     

Gammadelta (γδ) T lymphocytes do not impact the development of early atherosclerosis

Objective

Gammadelta (γδ) T cells are a subset of pro-inflammatory innate-like T lymphocytes that serve as a bridge between innate and adaptive immunity. γδ T cells are highly enriched in cholesterol compared to αβ T cells. In this study, we aimed to identify the role of γδ T cells in atherosclerosis, a cholesterol and inflammation-driven disease.

Methods

We found that the percentages of γδ T cells are increased in ApoE^−/− mice fed a Western diet. We generated TCRδ^−/−ApoE^−/− mice and fed them either rodent chow or a Western diet for ten weeks for the assessment of atherosclerosis.

Results

The atherosclerotic lesion size in diet-fed TCRδ^−/−ApoE^−/− mice was similar to that of diet-fed ApoE^−/− mice. There were no differences in cytokine production or numbers of αβ T cells in aorta of TCRδ^−/−ApoE^−/− mice. Plasma lipoprotein profiles were unchanged by the absence of γδ T cells.

Conclusion

Our data suggest that γδ T cells do not contribute to early atherosclerotic plaque development.     

Lack of vitamin D receptor causes stress-induced premature senescence in vascular smooth muscle cells through enhanced local angiotensin-II signals

Objectives

The inhibition of the renal renin-angiotensin system by the active form of vitamin D contributes to the cardiovascular health benefits of a normal vitamin D status. Local production of angiotensin-II in the vascular wall is a potent mediator of oxidative stress, prompting premature senescence. Herein, our objective was to examine the impact of defective vitamin D signalling on local angiotensin-II levels and arterial health.

Methods

Primary cultures of aortic vascular smooth muscle cells (VSMC) from wild-type and vitamin D receptor-knockout (VDRKO) mice were used for the assessment of cell growth, angiotensin-II and superoxide anion production and expression levels of cathepsin D, angiotensin-II type 1 receptor and p57^Kip2. The in vitro findings were confirmed histologically in aortas from wild-type and VDRKO mice.

Results

VSMC from VDRKO mice produced more angiotensin-II in culture, and elicited higher levels of cathepsin D, an enzyme with renin-like activity, and angiotensin-II type 1 receptor, than wild-type mice. Accordingly, VDRKO VSMC showed higher intracellular superoxide anion production, which could be suppressed by cathepsin D, angiotensin-II type 1 receptor or NADPH oxidase antagonists. VDRKO cells presented higher levels of p57^Kip2, impaired proliferation and premature senescence, all of them blunted upon inhibition of angiotensin-II signalling. In vivo studies confirmed higher levels of cathepsin D, angiotensin-II type 1 receptor and p57^Kip2 in aortas from VDRKO mice.

Conclusion

The beneficial effects of active vitamin D in vascular health could be a result of the attenuation of local production of angiotensin-II and downstream free radicals, thus preventing the premature senescence of VSMC.     

p53 negatively regulates the osteogenic differentiation of vascular smooth muscle cells in mice with chronic kidney disease

Aim

To investigate the osteogenic differentiation of vascular smooth muscle cells (VSMCs) in mice with chronic kidney disease (CKD) and to evaluate the effects of p53 on the osteogenic differentiation of the VSMCs.

Methods

Experimental models of CKD-associated vascular calcification generated by five-sixth (5/6) nephrectomy (Nx) and a high-phosphate (HP) diet were used in p53+/+ and p53–/– mice. Following 5/6 Nx, aortic calcification, markers of osteogenic differentiation, VSMCs and p53 protein in aortic tissues were studied.

Results

Aortic calcification was observed after eight weeks following 5/6 Nx in mice of both genotypes, and expression of the markers of osteogenic differentiation in the VSMCs was increased. These changes were continuously observed up to 12 weeks after 5/6 Nx, and particularly after 5/6 Nx + HP. Compared with p53+/+ mice, aortic calcification in p53–/– mice was more severe (p < 0.001). Expression of the markers of osteogenic differentiation was noticeably increased (p < 0.001), while expression of the marker of VSMCs had decreased (p < 0.001). Statistical analysis demonstrated that the markers of osteogenic differentiation were negatively correlated with p53, and the marker of VSMCs was positively correlated with p53 (p < 0.001).

Conclusion

p53 has the potential to negatively regulate the osteogenic differentiation of VSMCs in CKD mice.     

Protection of Endothelial Cells,Inhibition of Neointimal Hyperplasia by β-elemene in an Injured Artery

Aims  

It is generally accepted that the oxidative stress and the proliferative activity of vascular smooth muscle cells (VSMCs) contribute to the pathogenesis of neointimal hyperplasia after vascular injury. Although β-elemene (β-1-methyl-1-vinyl-2, 4-diisopropenyl-cyclohexane) has been used as an antitumour drug, its therapeutic effect on vascular diseases has not yet been determined. In this study, we investigated whether β-elemene could inhibit oxidative damage of vascular endothelial cells, suppress VSMCs growth and prevent neointimal hyperplasia.     

Targeted mouse complement inhibitor CR2-Crry protects against the development of atherosclerosis in mice

Objective

Atherosclerosis is a chronic inflammatory and immune vascular disease, and clinical and experimental evidence has indicated an important role of complement activation products, including the terminal membrane attack complex (MAC), in atherogenesis. Here, we investigated whether complement inhibition represents a potential therapeutic strategy to treat/prevent atherogenesis using CR2-Crry, a recently described complement inhibitor that specifically targets to sites of C3 activation.

Methods and results

Previous studies demonstrated that loss of CD59 (a membrane inhibitor of MAC formation) accelerated atherogenesis in Apoe deficient (Apoe^−/−) mice. Here, both CD59 sufficient and CD59 deficient mice in an Apoe deficient background (namely, mCd59 ab^+/+/Apoe^−/− and mCd59 ab^−/−/Apoe^−/−) were treated with CR2-Crry for 4 and 2 months respectively, while maintained on a high fat diet. Compared to control treatment, CR2-Crry treatment resulted in significantly fewer atherosclerotic lesions in the aorta and aortic root, and inhibited the accelerated atherogenesis seen in mCd59 ab^+/+/Apoe^−/− and mCd59 ab^−/−/Apoe^−/− mice. CR2-Crry treatment also resulted in significantly reduced C3 and MAC deposition in the vasculature of both mice, as well as a significant reduction in the number of infiltrating macrophages and T cells.

Conclusion

The data demonstrate the therapeutic potential of targeted complement inhibition.     

The influence of apoE-deficiency and LDL-receptor-deficiency on the HDL subpopulation profile in mice and in humans

Objective

As apoE^−/− and LDL-Receptor^−/− mice are commonly used in atherosclerosis research; our objective was to point out the differences in HDL metabolism between mice and humans regarding the roles of apoE and LDLR.

Methods

We examined HDL particles obtained from wild type (WT), LDLR^−/−, and apoE^−/− mice, as well as from normal, homozygous familial hypercholesterolemic (FH), and apoE-deficient human subjects by 2-dimensional non-denaturing PAGE followed by immunoblot and image analysis.

Results

In WT mice, the majority of apoA-I was in large (9.0–12.0 nm), α-mobility HDL with trace amounts of apoA-I in small, preβ-1 HDL. In LDL^−/− mice, both apoA-I- and apoE-containing HDL looked normal. About one-third of apoE was associated with large apoA-I-containing HDL (LpA-I:E) and two-thirds formed large HDL without apoA-I (LpE). In apoE^−/− mice, apoA-I was detected in multiple, β-preβ-mobility, tightly-packed bands (7.0–13.0 nm) indicating that apoA-I in these animals was present only in poorly-lipidated, discoidal particles. Neither FH nor apoE-deficient humans showed significant alterations in apoA-I-containing HDL particles as compared to non-carriers.

Conclusions

Our data indicate that apoE is necessary for the formation of spherical, lipidated HDL particles in mice, but not in humans, probably because mice lack CETP. Based on our data, we hypothesize that apoE^−/− mice have little or no functional HDL, therefore results from apoE^−/− mice cannot be extrapolated to humans without taking this significant difference into consideration.     

Role of bone mineral density in the inverse relationship between body size and aortic calcification: Results from the Baltimore Longitudinal Study of Aging

Objective

There is a J-shaped relationship between body mass index (BMI) and cardiovascular outcomes in elderly patients (obesity paradox). Whether low BMI correlates with aortic calcification (AC) and whether this association is accounted for by bone demineralization is uncertain.

Methods

Presence of AC was evaluated in 687 community-dwelling individuals (49% male, mean age 67 ± 13 years) using CT images of the thoracic, upper and lower abdominal aorta, and scored from 0 to 3 according to number of sites that showed any calcification. Whole-body bone mineral density (BMD) was evaluated by dual-energy X-ray absorptiometry. Predictors of AC were assessed by logistic regression, and the role of BMD using mediation analysis.

Results

Age and cardiovascular risk factors were positively associated while both BMI (r = −0.11, p < 0.01) and BMD (r = −0.17, p < 0.0001) were negatively associated with AC severity. In multivariate models, lower BMI (OR 0.96, 95%CI 0.92–0.99, p = 0.01), older age, higher systolic blood pressure, use of lipid-lowering drugs and smoking were independent predictors of AC. A nonlinear relationship between BMI and AC was noticed (p = 0.03), with decreased AC severity among overweight participants. After adjusting for BMD, the coefficient relating BMI to AC was reduced by 14% and was no longer significant, whereas BMD remained negatively associated with AC (OR 0.82, 95%CI 0.069–0.96, p = 0.01), with a trend for a stronger relationship in older participants.

Conclusion

Low BMI is associated with increased AC, possibly through calcium mobilization from bone, resulting in low BMD. Prevention of weight loss and bone demineralization with aging may help reducing AC.     

Levels of Circulating CD34/KDR Cells Do Not Predict Coronary In-Stent Restenosis

Background

Angiographic and clinical parameters are poor predictors of in-stent restenosis. Bone marrow–derived CD34⁺ cells that coexpress a receptor for vascular endothelial growth factor (kinase insert domain receptor [KDR]) are committed to endothelial lineage. Mobilization and infusion of CD34⁺/KDR⁺ cells accelerates re-endothelialization and reduces neointimal thickness in vascular injury models. Bioengineered stents capturing CD34⁺ cells also show expedited re-endothelialization. We examined whether circulating CD34⁺/KDR⁺ cell counts can be used to predict restenosis in a bare-metal stent (BMS).

Methods

CD34⁺/KDR⁺ cells were counted by flow cytometry in 124 nondiabetic patients before BMS implantation and the relation to in-stent late luminal loss (LLL) was examined by angiography at 6 months (primary end point). Neointima was also quantified as the maximum percentage area stenosis (M%AS) and percentage volume intima hyperplasia (%VIH) on intravascular ultrasonography (secondary end points).

Results

Multiple linear regression analysis, taking into account implanted stent length and diameter, revealed no relation between CD34⁺/KDR⁺ cell counts and LLL (partial regression coefficient b = 0.11; 95% confidence interval [CI], −0.19-0.42; P = 0.46). Similarly, no relation between CD34⁺/KDR⁺ cell counts and M%AS or %VIH could be demonstrated. Moreover, the increase in CD34⁺/KDR⁺ cell counts over 6 months was unrelated to LLL (b = −0.15; 95% CI, −0.42-0.12; P = 0.28), M%AS, and %VIH.

Conclusions

Although our study does not exclude a pathophysiologic role for CD34⁺/KDR⁺ cells in the formation of neointima, cell counts before percutaneous coronary intervention proved to be unrelated to LLL or intravascular ultrasonographically derived restenosis parameters in coronary BMSs at 6 months.