Atherosclerosis progression is a result of chronic and non-resolving inflammation, effective treatments for which still remain to be developed. We designed and developed branched poly(ß-amino ester) nanoparticles (NPs) containing plasmid DNA encoding IL-10, a potent anti-inflammatory cytokine to atherosclerosis. The NPs (NP-VHPK) are functionalized with a targeting peptide (VHPK) specific for VCAM-1, which is overexpressed by endothelial cells at sites of atherosclerotic plaque. The anionic coating affords NP-VHPK with significantly lower toxicity than uncoated NPs in both endothelial cells and red blood cells (RBCs). Following injection of NP-VHPK in ApoE−/− mice, Cy5-labelled IL-10 significantly accumulates in both whole aortas and aortic sinus sections containing plaque compared to injection with a non-targeted control. Furthermore, IL-10 gene delivery results in an attenuation of inflammation locally at the plaque site. NP-VHPK may thus have the potential to reduce the inflammatory component of atherosclerosis in a safe and effective manner. Atherosclerosis is a chronic inflammatory disease that results in the formation of lipid-laden plaques within vascular walls. Although treatments using drugs and antibodies are now beginning to address the inflammation in atherosclerosis, neither is sufficient for long-term therapy. In this paper, we introduce a strategy to deliver genes encoding the anti-inflammatory protein interleukin-10 (IL-10) in vivo. We showed that Branched Poly(ß-aminoester) carrying the IL-10 gene are able to localize specifically at the plaque via surface-functionalized targeting moieties against inflamed VCAM-1 and/or ICAM-1 and to facilitate gene transcription by ECs to increase the local concentration of the IL-10 within the plaque. To date, there is no report involving non-viral nanotechnology to provide gene-based therapies for atherosclerosis.
Arsenic is a widespread environmental contaminant to which millions of people are exposed worldwide. Exposure to arsenic is epidemiologically linked to increased cardiovascular disease, such as atherosclerosis. However, the effects of moderate concentrations of arsenic on atherosclerosis formation are unknown. Therefore, we utilized an in vivo ApoE−/− mouse model to assess the effects of chronic moderate exposure to arsenic on plaque formation and composition in order to facilitate mechanistic investigations. Mice exposed to 200 ppb arsenic developed atherosclerotic lesions, a lower exposure than previously reported. In addition, arsenic modified the plaque content, rendering them potentially less stable and consequently, potentially more dangerous. Moreover, we observed that the lower exposure concentration was more atherogenic than the higher concentration. Arsenic-enhanced lesions correlated with several proatherogenic molecular changes, including decreased liver X receptor (LXR) target gene expression and increased proinflammatory cytokines. Significantly, our observations suggest that chronic moderate arsenic exposure may be a greater cardiovascular health risk than previously anticipated.
Background: Inhibition of Four-and-a-half LIM domain protein-2 (FHL-2) attenuates atherosclerotic lesion formation and increases endothelial cell migration. Endothelial progenitor cells (EPCs) substantially contribute to endothelial repair. We investigated the role of FHL-2 in the regulation of early outgrowth EPC number and function. Methods and Results: Early outgrowth EPCs were obtained from human peripheral blood. FHL-2 knockdown in EPCs by small-interfering RNA (siRNA) resulted in a significant increase in EPC number and a reduction of apoptosis (by 40%), as indicated by a decrease of cleaved caspase-3, through activation and translocation to the membrane, of sphingosine kinase-1 (SK-1), enzyme that metabolizes sphingosine- 1 phosphate (s1p). Furthermore, FHL-2 siRNA increased significantly (2 fold) stromal derived factor (SDF) -1- induced EPC migration; through upregulation of α-v/β-3 and α-v/β-5 integrins; this was associated with an increase of the F-actin binding protein cortactin, known to promote migration. Interestingly, increased SDF-1- induced EPC migration and upregulation of cortactin by FHL-2 siRNA were totally prevented by CAY10621 , a specific inhibitor of SK-1. In addition stimulation of EPCs with exogenous s1p peptide significantly decreased apoptosis and increased SDF-1- induced migration. These results were confirmed In vivo using FHL-2 knockout (FHL-2 -/-) mice. Moreover, apoptosis was significantly decreased and migration increased in endothelial cells exposed to the conditioned medium of FHL-2 -/- vs. WT EPCs. These effects were abolished by VPC23019 , an antagonist of sphingosine- 1- phosphate receptor- 1 and 3. Finally, reendothelialization after focal carotid endothelial electric injury in WT mice was significantly increased after application of spleen-derived progenitor cells from FHL-2 -/- mice vs. WT mice. Conclusions: Our findings suggest that FHL-2 negatively regulates early outgrowth EPC function and secretion of paracrine factors. FHL-2 inhibition reduces apoptosis, enhances survival and migratory capacity of EPCs and ECs by upregulating SK-1/s1p pathway, integrin subunits and cortactin; which results in the improvement of endothelial regeneration.
Hypertension is associated with vascular remodeling characterized by rearrangement of extracellular matrix proteins. To evaluate how matrix metalloproteinase (MMP)-9 contributes to the progression of hypertensive vascular disease in vivo, wild-type (wt) or MMP-9 −/− mice were treated with angiotensin II (Ang II; 1 μg/kg per minute, by minipump) plus a 5% NaCl diet during 10 days. Baseline blood pressure was equivalent in wt and knockout mice, but Ang II treatment increased systolic blood pressure to a greater extent ( P <0.05) in MMP-9 −/− mice (94±6 to 134±6 mm Hg; P <0.001) than in wt animals (93±4 to 114±6 mm Hg; P <0.01). In wt mice, Ang II treatment increased the carotid artery pressure-diameter relationship significantly, and maximal diameter reached 981±19 μm ( P <0.01 versus sham; 891±10 μm). In contrast, in MMP-9 −/− mice, carotid artery compliance was actually reduced after Ang II ( P <0.05), and maximal diameter only reached 878±13 μm. Ang II treatment induced MMP-2 and increased carotid media thickness equally in both phenotypes. However, MMP-9 induction and in situ gelatinase activity were only enhanced in Ang II-treated wt mice, and vessels from these mice also produced more collagen I breakdown products than their MMP-9 −/− counterparts ( P <0.05). Inversely, staining for collagen IV was particularly enhanced in vessels from MMP-9 −/− mice treated with Ang II. These results demonstrate the following: (1) the onset of Ang II-induced hypertension is accompanied by increased MMP-9 activity in conductance vessels; (2) absence of MMP-9 activity results in vessel stiffness and increased pulse pressure; and (3) MMP-9 activation is associated with a beneficial role early on in hypertension by preserving vessel compliance and alleviating blood pressure increase.
Rationale: Inhibition of four-and-a-half LIM domain protein-2 (FHL2) attenuates atherosclerotic lesion formation and increases endothelial cell migration. Early outgrowth cells (EOCs) contribute substantially to endothelial repair. Objective: We investigated the role of FHL2 in the regulation of EOCs. Methods and Results: Human EOCs were cultured from peripheral blood. FHL2 knockdown in EOCs by siRNA resulted in increased EOC numbers and reduced apoptosis, as indicated by decreased cleaved caspase-III and reduced Bax/Bcl-2 expression ratio. This was mediated through increased phosphorylation and membrane translocation of sphingosine kinase-1, increased sphingosine-1-phosphate levels, and Akt phosphorylation. FHL2 knockdown increased stromal cell–derived factor-1–induced EOC migration through upregulation of αv/β3, αv/β5, and β2 integrins, associated with increased cortactin expression. Reduced apoptosis, increased EOC migration, and cortactin upregulation by FHL2 siRNA were prevented by CAY10621, the sphingosine kinase-1 inhibitor, and the sphingosine-1-phosphate receptor-1/-3 antagonist VPC23019. These findings were confirmed using spleen-derived EOCs from FHL2 −/− mice. Apoptosis was decreased and migration increased in endothelial cells exposed to the conditioned medium of FHL2 −/− versus wild-type (WT) EOCs. These paracrine effects were abolished by VPC23019. Importantly, reendothelialization after focal carotid endothelial injury in WT mice was significantly increased after intravenous injection of FHL2 −/− versus WT EOCs. Conclusions: Our findings suggest that FHL2 negatively regulates EOC survival, migration, and paracrine function. FHL2 inhibition in EOCs reduces apoptosis and enhances survival and migratory capacity of both EOCs and surrounding endothelial cells by activation of the sphingosine kinase-1/sphingosine-1-phosphate pathway, resulting in improvement of endothelial regeneration.
Background: Four-and-a-half LIM domain protein-2 (FHL2) is expressed in endothelial and vascular smooth muscle cells. It negatively regulates endothelial cell survival and migration, but its role in atherogenesis is unknown. Methods and Results: FHL2-deficient (FHL2-/-) mice were crossed with apolipoprotein E-deficient (ApoE-/-) mice to generate ApoE/FHL2-/- mice. After high-fat, high-cholesterol diet, ApoE/FHL2-/- mice displayed significantly smaller atherosclerotic plaques than ApoE-/- mice in the aortic sinus, the brachiocephalic artery and the aorta. This was associated with significantly enhanced collagen and smooth muscle cell contents and a significant 2-fold reduction of macrophage content within the plaques of ApoE/FHL-2-/- vs ApoE-/- mice. There was a significant reduction in aortic ICAM-1 mRNA and VCAM-1 protein expression in the plaques of ApoE/FHL2-/- mice. Aortic gene expression of CX3CL1 and CCL5 was significantly increased in ApoE/FHL2-/- vs ApoE-/- mice. Peritoneal thioglycollate injection elicited equivalent numbers of monocytes and macrophages in both groups, but a significantly lower number of pro-inflammatory Ly6C-high monocytes were recruited in ApoE/FHL2-/- vs ApoE-/- mice. Furthermore, mRNA levels of CX3CR1 were 2-fold higher in monocytes from ApoE/FHL2-/- vs ApoE-/- mice. Finally, we investigated the potential importance of myeloid cell FHL2 deficiency in atherosclerosis. After being irradiated, ApoE-/- or ApoE/FHL2-/- mice were transplanted with ApoE-/- or ApoE/FHL2-/- bone marrow. After high-fat, high-cholesterol diet, both chimeric groups developed significantly smaller plaques than ApoE-/- mice transplanted with ApoE-/- bone marrow. Conclusion: These results suggest that FHL2 in both myeloid and vascular cells may play an important role in atherogenesis by promoting pro-inflammatory chemokine production, adhesion molecule expression, and pro-inflammatory monocyte recruitment.
Background: Four-and-a-half LIM domain protein 2 (FHL2) is an adaptor molecule that regulates signalling cascades and gene transcription. We have uncovered vasculoprotective and atheroprotective effects of FHL2 knockout in mice. Since B cells could regulate these processes, we investigated the potential role of FHL2 in B cell function and activity. Methods and Results: Under basal conditions, FHL2-/- mice presented a mild splenomegaly (84.0±9.0 vs 68.0±5.0mg), associated with bigger spleen follicles (1.6 fold) and higher proportions of spleen B cells (56.0±2.0 vs 40.0±9.0%) vs WT mice. Flow cytometry confirmed significantly higher (p<0.05) proportions of B cells in the follicles (Fo)(CD23+CD21lo/int) (77.0±1.0 vs 67.0±3.0%) and the marginal zone (Mz)(CD23-,CD21+)(7.0±1.0 vs 2.5±1.0%) of FHL2-/- vs WT mice. Mice were injected with sheep red blood cells (SRBC) to elicit a T cell-dependent B cell activation. SRBC did not influence Fo and Mz B cell numbers, but it did affect germinal center (GC) formation. GCs are zones within Fo where T cells activate B cells to produce high affinity antibodies. SRBC induced a 5- and 2.5-fold increase in B cells (GL7+) within FHL2-/- and WT GCs, respectively. However, there was no increase in plasma IgG1 levels in FHL2-/- mice in response to SRBC, whereas a 3-fold increase was observed in WT mice (1158±227 vs 2943±226ng/ml; p<0.05). Instead, plasma IgM levels were higher in FHL2-/- than WT SRBC mice. Moreover, SRBC only induced a modest, 1.3-fold fold increase in Fo T helper cells (CXCR5+PD1+) in FHL2-/- compared with a 6-fold increase in WT. Nevertheless, FHL2-/- B cells successfully proliferated and underwent class switch recombination in response to LPS, anti-CD40 and IL-4 in vitro, indicating that these cells were not defective. Conclusion: These results suggest that FHL2 plays a critical role in spleen antibody production in response to a T-dependent antigen, possibly by affecting activation or antigen presentation by Fo T helper cells.
Arsenic is metabolized through a series of oxidative methylation reactions by arsenic (3) methyltransferase (As3MT) to yield methylated intermediates. Although arsenic exposure is known to increase the risk of atherosclerosis, the contribution of arsenic methylation and As3MT remains undefined.Our objective was to define whether methylated arsenic intermediates were proatherogenic and whether arsenic biotransformation by As3MT was required for arsenic-enhanced atherosclerosis.We utilized the apoE−/− mouse model to compare atherosclerotic plaque size and composition after inorganic arsenic, methylated arsenical, or arsenobetaine exposure in drinking water. We also generated apoE−/−/As3mt−/− double knockout mice to test whether As3MT-mediated biotransformation was required for the proatherogenic effects of inorganic arsenite. Furthermore, As3MT expression and function were assessed in in vitro cultures of plaque-resident cells. Finally, bone marrow transplantation studies were performed to define the contribution of As3MT-mediated methylation in different cell types to the development of atherosclerosis after inorganic arsenic exposure.We found that methylated arsenicals, but not arsenobetaine, are proatherogenic and that As3MT is required for arsenic to induce reactive oxygen species and promote atherosclerosis. Importantly, As3MT was expressed and functional in multiple plaque-resident cell types, and transplant studies indicated that As3MT is required in extrahepatic tissues to promote atherosclerosis.Taken together, our findings indicate that As3MT acts to promote cardiovascular toxicity of arsenic and suggest that human AS3MT SNPs that correlate with enzyme function could predict those most at risk to develop atherosclerosis among the millions that are exposed to arsenic. https://doi.org/10.1289/EHP806.
