Accelerated atherosclerosis in beta-thalassemia.

Children with beta-thalassemia (BT) present with an increase in carotid intima-medial thickness, an early sign suggestive of premature atherosclerosis. However, it is unknown if there is a direct relationship between BT and atherosclerotic disease. To evaluate this, wild-type (WT, littermates) and BT (Hbbth3/+) mice, both male and female, were placed on a 3-mo high-fat diet with low-density lipoprotein receptor suppression via overexpression of proprotein convertase subtilisin/kexin type 9 (PCSK9) gain-of-function mutation (D377Y). Mechanistically, we hypothesize that heme-mediated oxidative stress creates a proatherogenic environment in BT because BT is a hemolytic anemia that has increased free heme and exhausted hemopexin, heme's endogenous scavenger, in the vasculature. We evaluated the effect of hemopexin (HPX) therapy, mediated via an adeno-associated virus, to the progression of atherosclerosis in BT and a phenylhydrazine-induced model of intravascular hemolysis. In addition, we evaluated the effect of deferiprone (DFP)-mediated iron chelation in the progression of atherosclerosis in BT mice. Aortic en face and aortic root lesion area analysis revealed elevated plaque accumulation in both male and female BT mice compared with WT mice. Hemopexin therapy was able to decrease plaque accumulation in both BT mice and mice on our phenylhydrazine (PHZ)-induced model of hemolysis. DFP decreased atherosclerosis in BT mice but did not provide an additive benefit to HPX therapy. Our data demonstrate for the first time that the underlying pathophysiology of BT leads to accelerated atherosclerosis and shows that heme contributes to atherosclerotic plaque development in BT.NEW & NOTEWORTHY This work definitively shows for the first time that beta-thalassemia leads to accelerated atherosclerosis. We demonstrated that intravascular hemolysis is a prominent feature in beta-thalassemia and the resulting increases in free heme are mechanistically relevant. Adeno-associated virus (AAV)-hemopexin therapy led to decreased free heme and atherosclerotic plaque area in both beta-thalassemia and phenylhydrazine-treated mice. Deferiprone-mediated iron chelation led to deceased plaque accumulation in beta-thalassemia mice but provided no additive benefit to hemopexin therapy.

Increasing nitric oxide bioavailability fails to improve collateral vessel formation in humanized sickle cell mice.

Sickle cell disease (SCD) is associated with repeated bouts of vascular insufficiency leading to organ dysfunction. Deficits in revascularization following vascular injury are evident in SCD patients and animal models. We aimed to elucidate whether enhancing nitric oxide bioavailability in SCD mice improves outcomes in a model of vascular insufficiency. Townes AA (wild type) and SS (sickle cell) mice were treated with either L-Arginine (5% in drinking water), L-NAME (N(ω)-nitro-L-arginine methyl ester; 1 g/L in drinking water) or NO-generating hydrogel (PA-YK-NO), then subjected to hindlimb ischemia via femoral artery ligation and excision. Perfusion recovery was monitored over 28 days via LASER Doppler perfusion imaging. Consistent with previous findings, perfusion was impaired in SS mice (63 ± 4% of non-ischemic limb perfusion in AA vs 33 ± 3% in SS; day 28; P < 0.001; n = 5-7) and associated with increased necrosis. L-Arginine treatment had no significant effect on perfusion recovery or necrosis (n = 5-7). PA-YK-NO treatment led to worsened perfusion recovery (19 ± 3 vs. 32 ± 3 in vehicle-treated mice; day 7; P < 0.05; n = 4-5), increased necrosis score (P < 0.05, n = 4-5) and a 46% increase in hindlimb peroxynitrite (P = 0.055, n = 4-5). Interestingly, L-NAME worsened outcomes in SS mice with decreased in vivo lectin staining following ischemia (7 ± 2% area in untreated vs 4 ± 2% in treated mice, P < 0.05, n = 5). Our findings demonstrate that L-arginine and direct NO delivery both fail to improve postischemic neovascularization in SCD. Addition of NO to the inflammatory, oxidative environment in SCD may result in further oxidative stress and limit recovery.

Osteopontin isoforms differentially promote arteriogenesis in response to ischemia via macrophage accumulation and survival.

Osteopontin (OPN) is critical for ischemia-induced neovascularization. Unlike rodents, humans express three OPN isoforms (a, b, and c); however, the roles of these isoforms in post-ischemic neovascularization and cell migration remain undefined. Our objective was to determine if OPN isoforms differentially affect post-ischemic neovascularization and to elucidate the mechanisms underlying these differences. To investigate if human OPN isoforms exert divergent effects on post-ischemic neovascularization, we utilized OPN-/- mice and a loss-of-function/gain-of-function approach in vivo and in vitro. In this study OPN-/- mice underwent hindlimb ischemia surgery and 1.5 × 106 lentivirus particles were administered intramuscularly to overexpress OPNa, OPNb, or OPNc. OPNa and OPNc significantly improved limb perfusion 30.4% ± 0.8 and 70.9% ± 6.3, respectively, and this translated to improved functional limb use, as measured by voluntary running wheel utilization. OPNa- and OPNc-treated animals exhibited significant increases in arteriogenesis, defined here as the remodeling of existing arterioles into larger conductance arteries. Macrophages play a prominent role in the arteriogenesis process and OPNa- and OPNc-treated animals showed significant increases in macrophage accumulation in vivo. In vitro, OPN isoforms did not affect macrophage polarization, whereas all three isoforms increased macrophage survival and decreased macrophage apoptosis. However, OPN isoforms exert differential effects on macrophage migration, where OPNa and OPNc significantly increased macrophage migration, with OPNc serving as the most potent isoform. In conclusion, human OPN isoforms exert divergent effects on neovascularization through differential effects on arteriogenesis and macrophage accumulation in vivo and on macrophage migration and survival, but not polarization, in vitro. Altogether, these data support that human OPN isoforms may represent novel therapeutic targets to improve neovascualrization and preserve tissue function in patients with obstructive artery diseases.

Poldip2 knockdown inhibits vascular smooth muscle proliferation and neointima formation by regulating the expression of PCNA and p21.

Polymerase delta-interacting protein 2 (Poldip2) is a multi-functional protein with numerous roles in the vasculature, including the regulation of cell apoptosis and migration, as well as extracellular matrix deposition; however, its role in VSMC proliferation and neointimal formation is unknown. In this study, we investigated the role of Poldip2 in intraluminal wire-injury induced neointima formation and proliferation of vascular smooth muscle cells in vitro and in vivo. Poldip2 expression was observed in the intima and media of human atherosclerotic arteries, where it colocalized with proliferating cell nuclear antigen (PCNA). Wire injury of femoral arteries of Poldip2+/+ mice induced robust neointimal formation after 2 weeks, which was impaired in Poldip2+/‒ mice. PCNA expression was significantly reduced and expression of the cell cycle inhibitor p21 was significantly increased in wire-injured arteries of Poldip2+/‒ animals compared to wild-type controls. No difference was observed in apoptosis. Downregulation of Poldip2 in rat aortic smooth muscle cells significantly reduced serum-induced proliferation and PCNA expression, but upregulated p21 expression. Downregulation of p21 using siRNA reversed the inhibition of proliferation induced by knockdown of Poldip2. These results indicate that Poldip2 plays a critical role in the proliferation of VSMCs.

Impaired Collateral Vessel Formation in Sickle Cell Disease.

OBJECTIVE: The adaptive response to vascular injury is the formation of functional collateral vessels to maintain organ integrity. Many of the clinical complications associated with sickle cell disease can be attributed to repeated bouts of vascular insufficiency, yet the detailed mechanisms of collateral vessel formation after injury are largely unknown in sickle cell disease. Here, we characterize postischemic neovascularization in sickle cell disease and the role of neutrophils in the production of reactive oxygen species. APPROACH AND RESULTS: We induced hindlimb ischemia by ligation of the femoral artery in Townes SS (sickle cell) mice compared with AA (wild type) mice. Perfusion recovery, ascertained using LASER (light amplification by stimulated emission of radiation) Doppler perfusion imaging, showed significant diminution in collateral vessel formation in SS mice after hindlimb ischemia (76±13% AA versus 34±10% in SS by day 28; P<0.001; n=10 per group). The incidence of amputation (25% versus 5%) and foot necrosis (80% versus 15%) after hindlimb ischemia was significantly increased in the SS mice. Motor function recovery evaluation by the running wheel assay was also impaired in SS mice (36% versus 97% at 28 days post-hindlimb ischemia; P<0.001). This phenotype was associated with persistent and excessive production of reactive oxygen species by neutrophils. Importantly, neutrophil depletion or treatment with the antioxidant N-acetylcysteine reduced oxidative stress and improved functional collateral formation in the SS mice. CONCLUSIONS: Our data suggest dysfunctional collateral vessel formation in SS mice after vascular injury and provide a mechanistic basis for the multiple vascular complications of sickle cell disease.

The receptor for advanced glycation end products impairs collateral formation in both diabetic and non-diabetic mice.

Diabetics often have poor perfusion in their limbs as a result of peripheral artery disease and an impaired ability to generate collateral vessels. The receptor for advanced glycation end products (RAGE) is one protein that is thought to play a detrimental role in collateral development in diabetics due to increased levels of advanced glycation end products (AGE), one of its ligands, in diabetes. Thus, the aim of this study was to investigate the role of RAGE in both diabetic and non-diabetic settings in a model of collateral formation in mice. Streptozotocin was used to induce diabetes in both wild type and RAGE knockout mice. Increased levels of the AGE, Nɛ-(carboxymethyl) lysine (CML), were confirmed via an ELISA. A hindlimb ischemia model, in which the femoral artery is ligated, was used to drive collateral growth and reperfusion was assessed using laser Doppler perfusion imaging and histological analysis of vessels in the muscle. Both of these measurements showed impaired collateral growth in diabetic compared with wild-type mice as well as improved collateral growth in both diabetic and non-diabetic RAGE knockout mice when compared their wild-type counterparts. Distance on a freely accessed running wheel, used as a measure of perfusion recovery, showed that wild-type diabetic mice had functionally impaired recovery compared with their wild-type counterparts. Immunohistochemistry and immunoblotting showed that HMGB-1 (high-mobility group box 1), another RAGE ligand, was increased in the ischemic leg compared with the non-ischemic leg in all mice. This increase in HMGB-1 may explain improvement in animals lacking RAGE and its subsequent signaling. In conclusion, this study shows that RAGE impairs collateral growth in a diabetic setting and also in a non-diabetic setting. This demonstrates the importance of RAGE and alternate RAGE ligands in the setting of collateral vessel growth.

Polymerase δ-interacting protein 2 promotes postischemic neovascularization of the mouse hindlimb.

OBJECTIVE: Collateral vessel formation can functionally compensate for obstructive vascular lesions in patients with atherosclerosis. Neovascularization processes are triggered by fluid shear stress, hypoxia, growth factors, chemokines, proteases, and inflammation, as well as reactive oxygen species, in response to ischemia. Polymerase δ-interacting protein 2 (Poldip2) is a multifunctional protein that regulates focal adhesion turnover and vascular smooth muscle cell migration and modifies extracellular matrix composition. We, therefore, tested the hypothesis that loss of Poldip2 impairs collateral formation. APPROACH AND RESULTS: The mouse hindlimb ischemia model has been used to understand mechanisms involved in postnatal blood vessel formation. Poldip2(+/-) mice were subjected to femoral artery excision, and functional and morphological analysis of blood vessel formation was performed after injury. Heterozygous deletion of Poldip2 decreased the blood flow recovery and spontaneous running activity at 21 days after injury. H2O2 production, as well as the activity of matrix metalloproteinases-2 and -9, was reduced in these animals compared with Poldip2(+/+) mice. Infiltration of macrophages in the peri-injury muscle was also decreased; however, macrophage phenotype was similar between genotypes. In addition, the formation of capillaries and arterioles was impaired, as was angiogenesis, in agreement with a decrease in proliferation observed in endothelial cells treated with small interfering RNA against Poldip2. Finally, regression of newly formed vessels and apoptosis was more pronounced in Poldip2(+/-) mice. CONCLUSIONS: Together, these results suggest that Poldip2 promotes ischemia-induced collateral vessel formation via multiple mechanisms that likely involve reactive oxygen species-dependent activation of matrix metalloproteinase activity, as well as enhanced vascular cell growth and survival.

Overexpression of catalase in vascular smooth muscle cells prevents the formation of abdominal aortic aneurysms.

OBJECTIVE: Elevated levels of oxidative stress have been reported in abdominal aortic aneurysms (AAA), but which reactive oxygen species promotes the development of AAA remains unclear. Here, we investigate the effect of hydrogen peroxide (H2O2)-degrading enzyme catalase on the formation of AAA. APPROACH AND RESULTS: AAA were induced with the application of calcium chloride (CaCl2) on mouse infrarenal aortas. The administration of PEG-catalase, but not saline, attenuated the loss of tunica media and protected against AAA formation (0.91 ± 0.1 versus 0.76 ± 0.09 mm). Similarly, in a transgenic mouse model, catalase overexpression in the vascular smooth muscle cells preserved the thickness of tunica media and inhibited aortic dilatation by 50% (0.85 ± 0.14 versus 0.57 ± 0.08 mm). Further studies showed that injury with CaCl2 decreased catalase expression and activity in the aortic wall. Pharmacological administration or genetic overexpression of catalase restored catalase activity and subsequently decreased matrix metalloproteinase activity. In addition, a profound reduction in inflammatory markers and vascular smooth muscle cell apoptosis was evident in aortas of catalase-overexpressing mice. Interestingly, as opposed to infusion of PEG-catalase, chronic overexpression of catalase in vascular smooth muscle cells did not alter the total aortic H2O2 levels. CONCLUSIONS: The data suggest that a reduction in aortic wall catalase activity can predispose to AAA formation. Restoration of catalase activity in the vascular wall enhances aortic vascular smooth muscle cell survival and prevents AAA formation primarily through modulation of matrix metalloproteinase activity.

mRNA-binding protein ZFP36 is expressed in atherosclerotic lesions and reduces inflammation in aortic endothelial cells.

OBJECTIVE: We studied the expression and function of an mRNA-binding protein, zinc finger protein-36 (ZFP36), in vascular endothelial cells in vivo and in vitro. We tested the hypotheses that ZFP36 regulates inflammation in vascular endothelial cells and that it functions through direct binding to target cytokine mRNAs. We also tested whether ZFP36 inhibits nuclear factor-κB-mediated transcriptional responses in vascular endothelial cells. APPROACH AND RESULTS: ZFP36 was minimally expressed in healthy aorta but was expressed in endothelial cells overlying atherosclerotic lesions in mice and humans. The protein was also expressed in macrophage foam cells of atherosclerosis. ZFP36 was expressed in human aortic endothelial cells in response to bacterial lipopolysaccharide, glucocorticoid, and forskolin, but not oxidized low-density lipoproteins or angiotensin II. Functional studies demonstrated that ZFP36 reduces the expression of inflammatory cytokines in target cells by 2 distinct mechanisms: ZFP36 inhibits nuclear factor-κB transcriptional activation and also binds to cytokine mRNAs, leading to reduced transcript stability. CONCLUSIONS: ZFP36 is expressed in vascular endothelial cells and macrophage foam cells where it inhibits the expression of proinflammatory mRNA transcripts. The anti-inflammatory effects of ZFP36 in endothelial cells occur via both transcriptional and posttranscriptional mechanisms. Our data suggest that enhancing vascular ZFP36 expression might reduce vascular inflammation.

Polymerase delta interacting protein 2 sustains vascular structure and function.

OBJECTIVE: On the basis of previous evidence that polymerase delta interacting protein 2 (Poldip2) increases reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (Nox4) activity in vascular smooth muscle cells, we hypothesized that in vivo knockdown of Poldip2 would inhibit reactive oxygen species production and alter vascular function. APPROACH AND RESULTS: Because homozygous Poldip2 deletion is lethal, Poldip2(+/-) mice were used. Poldip2 mRNA and protein levels were reduced by ≈50% in Poldip2(+/-) aorta, with no change in p22phox, Nox1, Nox2, and Nox4 mRNAs. NADPH oxidase activity was also inhibited in Poldip2(+/-) tissue. Isolated aortas from Poldip2(+/-) mice demonstrated impaired phenylephrine and potassium chloride-induced contractions, increased stiffness, and reduced compliance associated with disruption of elastic lamellae and excessive extracellular matrix deposition. Collagen I secretion was elevated in cultured vascular smooth muscle cells from Poldip2(+/-) mice and restored by H2O2 supplementation, suggesting that this novel function of Poldip2 is mediated by reactive oxygen species. Furthermore, Poldip2(+/-) mice were protected against aortic dilatation in a model of experimental aneurysm, an effect consistent with increased collagen secretion. CONCLUSIONS: Poldip2 knockdown reduces H2O2 production in vivo, leading to increases in extracellular matrix, greater vascular stiffness, and impaired agonist-mediated contraction. Thus, unaltered expression of Poldip2 is necessary for vascular integrity and function.

The Role of Fatty Acid Synthase in the Vascular Smooth Muscle Cell to Foam Cell Transition.

Vascular smooth muscle cells (VSMCs), in their contractile and differentiated state, are fundamental for maintaining vascular function. Upon exposure to cholesterol (CHO), VSMCs undergo dedifferentiation, adopting characteristics of foam cells-lipid-laden, macrophage-like cells pivotal in atherosclerotic plaque formation. CHO uptake by VSMCs leads to two primary pathways: ABCA1-mediated efflux or storage in lipid droplets as cholesterol esters (CEs). CE formation, involving the condensation of free CHO and fatty acids, is catalyzed by sterol O-acyltransferase 1 (SOAT1). The necessary fatty acids are synthesized by the lipogenic enzyme fatty acid synthase (FASN), which we found to be upregulated in atherosclerotic human coronary arteries. This observation led us to hypothesize that FASN-mediated fatty acid biosynthesis is crucial in the transformation of VSMCs into foam cells. Our study reveals that CHO treatment upregulates FASN in human aortic SMCs, concurrent with increased expression of CD68 and upregulation of KLF4, markers associated with the foam cell transition. Crucially, downregulation of FASN inhibits the CHO-induced upregulation of CD68 and KLF4 in VSMCs. Additionally, FASN-deficient VSMCs exhibit hindered lipid accumulation and an impaired transition to the foam cell phenotype following CHO exposure, while the addition of the fatty acid palmitate, the main FASN product, exacerbates this transition. FASN-deficient cells also show decreased SOAT1 expression and elevated ABCA1. Notably, similar effects are observed in KLF4-deficient cells. Our findings demonstrate that FASN plays an essential role in the CHO-induced upregulation of KLF4 and the VSMC to foam cell transition and suggest that targeting FASN could be a novel therapeutic strategy to regulate VSMC phenotypic modulation.

Magnetic targeting of human mesenchymal stem cells with internalized superparamagnetic iron oxide nanoparticles.

Cell therapies offer exciting new opportunities for effectively treating many human diseases. However, delivery of therapeutic cells by intravenous injection, while convenient, relies on the relatively inefficient process of homing of cells to sites of injury. To address this limitation, a novel strategy has been developed to load cells with superparamagnetic iron oxide nanoparticles (SPIOs), and to attract them to specific sites within the body by applying an external magnetic field. The feasibility of this approach is demonstrated using human mesenchymal stem cells (hMSCs), which may have a significant potential for regenerative cell therapies due to their ease of isolation from autologous tissues, and their ability to differentiate into various lineages and modulate their paracrine activity in response to the microenvironment. The efficient loading of hMSCs with polyethylene glycol-coated SPIOs is achieved, and it is found that SPIOs are localized primarily in secondary lysosomes of hMSCs and are not toxic to the cells. Further, the key stem cell characteristics, including the immunophenotype of hMSCs and their ability to differentiate, are not altered by SPIO loading. Through both experimentation and mathematical modeling, it is shown that, under applied magnetic field gradients, SPIO-containing cells can be localized both in vitro and in vivo. The results suggest that, by loading SPIOs into hMSCs and applying appropriate magnetic field gradients, it is possible to target hMSCs to particular vascular networks.

Reactive oxygen species regulate osteopontin expression in a murine model of postischemic neovascularization.

OBJECTIVE: Previous findings from our laboratory demonstrated that neovascularization was impaired in osteopontin (OPN) knockout animals. However, the mechanisms responsible for the regulation of OPN expression in the setting of ischemia remain undefined. Therefore, we sought to determine whether OPN is upregulated in response to ischemia and hypothesized that hydrogen peroxide (H(2)O(2)) is a critical component of the signaling mechanism by which OPN expression is upregulated in response to ischemia in vivo. METHODS AND RESULTS: To determine whether ischemic injury upregulates OPN, we used a murine model of hindlimb ischemia. Femoral artery ligation in C57BL/6 mice significantly increased OPN expression and H(2)O(2) production. Infusion of C57BL/6 mice with polyethylene glycol-catalase (10 000 U/kg per day) or the use of transgenic mice with smooth muscle cell-specific catalase overexpression blunted ischemia-induced OPN, suggesting ischemia-induced OPN expression is H(2)O(2)-dependent. Decreased H(2)O(2)-mediated OPN blunted reperfusion and collateral formation in vivo. In contrast, the overexpression of OPN using lentivirus restored neovascularization. CONCLUSIONS: Scavenging H(2)O(2) blocks ischemia-induced OPN expression, providing evidence that ischemia-induced OPN expression is H(2)O(2) dependent. Decreased OPN expression impaired neovascularization, whereas overexpression of OPN increased angiogenesis, supporting our hypothesis that OPN is a critical mediator of postischemic neovascularization and a potential novel therapeutic target for inducing new vessel growth.

Folate receptor-targeted antioxidant therapy ameliorates renal ischemia-reperfusion injury.

Antioxidant therapy can protect against ischemic injury, but the inability to selectively target the kidney would require extremely high doses to achieve effective local concentrations of drug. Here, we developed a directed therapeutic that specifically targets an antioxidant to renal proximal tubule cells via the folate receptor. Because a local increase in superoxide contributes to renal ischemic injury, we created the folate-antioxidant conjugate 4-hydroxy-Tempo (tempol)-folate to target folate receptors, which are highly expressed in the proximal tubule. Dihydroethidium high-performance liquid chromatography demonstrated that conjugated tempol retained its efficacy to scavenge superoxide in proximal tubule cells. In a mouse model of renal ischemia-reperfusion injury, tempol-folate reduced renal superoxide levels more effectively than tempol alone. Furthermore, electron spin resonance revealed the successful targeting of the tempol-folate conjugate to the kidney and other tissues expressing folate receptors. Administration of tempol-folate protected the renal function of mice after ischemia-reperfusion injury and inhibited infiltration of macrophages. In conclusion, kidney-specific targeting of an antioxidant has therapeutic potential to prevent renal ischemic injury. Conjugation of other pharmaceuticals to folate may also facilitate the development of treatments for other kidney diseases.

Intestinal barrier dysfunction in murine sickle cell disease is associated with small intestine neutrophilic inflammation, oxidative stress, and dysbiosis.

The intestinal microbiome has emerged as a potential contributor to the severity of sickle cell disease (SCD). We sought to determine whether SCD mice exhibit intestinal barrier dysfunction, inflammation, and dysbiosis. Using the Townes humanized sickle cell mouse model, we found a 3-fold increase in intestinal permeability as assessed via FITC-dextran (4 kDa) assay in SS (SCD) mice compared to AA (wild type) mice (n = 4, p < 0.05). This was associated with 25 to 50% decreases in claudin-1, 3, and 15 and zonula occludens-1 gene expression (n = 8-10, p < 0.05) in the small intestine. Increased Ly6G staining demonstrated more neutrophils in the SS small intestine (3-fold, n = 5, p < 0.05) associated with increased expression of TNFα, IL-17A, CXCL1, and CD68 (2.5 to 5-fold, n = 7-10, p < 0.05). In addition, we observed 30 to 55% decreases in superoxide dismutase-1, glutathione peroxidase-1, and catalase antioxidant enzyme expression (n = 7-8, p < 0.05) concomitant to an increase in superoxide (2-fold, n = 4, p < 0.05). Importantly, all significant observations of a leaky gut phenotype and inflammation were limited to the small intestine and not observed in the colon. Finally, characterization of the composition of the microbiome within the small intestine revealed dysbiosis in SS mice compared to their AA littermates with 47 phyla to species-level significant alterations in amplicon sequence variants. We conclude that the intestinal barrier is compromised in SCD, associated with decreased gene expression of tight junction proteins, enhanced inflammation, oxidative stress, and gut microbiome dysbiosis, all specific to the small intestine.

The role of lysyl oxidase family members in the stabilization of abdominal aortic aneurysms.

Abdominal aortic aneurysms (AAAs) are a major cause of morbidity and mortality in the United States today. We employed a model for AAA development using apolipoprotein E knock out mice fed a high-fat diet and treated with ANG II and ß-aminopropionitrile (ß-APN) for 4 wk. ANG II induces hypertension and atherosclerotic disease, whereas ß-APN inhibits the activity of the lysyl oxidase/ lysyl oxidase-like protein (LOX/LOXL) family members. LOX/LOXL family members crosslink collagen and elastin in the extracellular matrix and therefore contribute to the integrity and stabilization of a healthy vessel wall. In this model, cotreatment with ANG II and ß-APN caused a 90% AAA incidence and increased atherosclerotic lesion formation from less than 5% to greater than 25% after 4 wk. In more atheroprotected mouse strains (C57BL/6 and BalbC), cotreatment with ANG II and ß-APN caused 50% and 40% AAA incidence, respectively. These data demonstrate the importance of LOX/LOXL to the stability of the vessel wall. Therapeutic strategies to overexpress LOX/LOXL enzymes or to support the crosslinking of soluble matrix proteins in a polymeric scaffold are a promising opportunity to achieve stabilization of AAAs.

Growth and regression of vasculature in healthy and diabetic mice after hindlimb ischemia.

The formation of vascular networks during embryogenesis and early stages of development encompasses complex and tightly regulated growth of blood vessels, followed by maturation of some vessels, and spatially controlled disconnection and pruning of others. The adult vasculature, while more quiescent, is also capable of adapting to changing physiological conditions by remodeling blood vessels. Numerous studies have focused on understanding key factors that drive vessel growth in the adult in response to ischemic injury. However, little is known about the extent of vessel rarefaction and its potential contribution to the final outcome of vascular recovery. We addressed this topic by characterizing the endogenous phases of vascular repair in a mouse model of hindlimb ischemia. We showed that this process is biphasic. It encompasses an initial rapid phase of vessel growth, followed by a later phase of vessel rarefaction. In healthy mice, this process resulted in partial recovery of perfusion and completely restored the ability of mice to run voluntarily. Given that the ability to revascularize can be compromised by a cardiovascular risk factor such as diabetes, we also examined vascular repair in diabetic mice. We found that paradoxically both the initial growth and subsequent regression of collateral vessels were more pronounced in the setting of diabetes and resulted in impaired recovery of perfusion and impaired functional status. In conclusion, our findings demonstrate that the formation of functional collateral vessels in the hindlimb requires vessel growth and subsequent vessel rarefaction. In the setting of diabetes, the physiological defect was not in the initial formation of vessels but rather in the inability to sustain newly formed vessels.

Overexpression of catalase in myeloid cells causes impaired postischemic neovascularization.

OBJECTIVE: Myeloid lineage cells (MLCs) such as macrophages are known to play a key role in postischemic neovascularization. However, the role of MLC-derived reactive oxygen species in this process and their specific chemical identity remain unknown. METHODS AND RESULTS: Transgenic mice with MLC-specific overexpression of catalase (Tg(Cat-MLC) mice) were created on a C57BL/6 background. Macrophage catalase activity was increased 3.4-fold compared with wild-type mice. After femoral artery ligation, laser Doppler perfusion imaging revealed impaired perfusion recovery in Tg(Cat-MLC) mice. This was associated with fewer collateral vessels, as assessed by microcomputed tomography angiography, and decreased capillary density. Impaired functional recovery of the ischemic limb was also evidenced by a 50% reduction in spontaneous running activity. The deficient neovascularization was associated with a blunted inflammatory response, characterized by decreased macrophage infiltration of ischemic tissues, and lower mRNA levels of inflammatory markers, such as tumor necrosis factor-α, osteopontin, and matrix mettaloproteinase-9. In vitro macrophage migration was impaired in Tg(Cat-MLC) mice, suggesting a role for H(2)O(2) in regulating the ability of macrophages to infiltrate ischemic tissues. CONCLUSIONS: MLC-derived H(2)O(2) plays a key role in promoting neovascularization in response to ischemia and is a necessary factor for the development of ischemia-induced inflammation.

Endothelial Poldip2 regulates sepsis-induced lung injury via Rho pathway activation.

AIMS: Sepsis-induced lung injury is associated with significant morbidity and mortality. Previously, we showed that heterozygous deletion of polymerase δ-interacting protein 2 (Poldip2) was protective against sepsis-induced lung injury. Since endothelial barrier disruption is thought to be the main mechanism of sepsis-induced lung injury, we sought to determine if the observed protection was specifically due to the effect of reduced endothelial Poldip2. METHODS AND RESULTS: Endothelial-specific Poldip2 knock-out mice (EC-/-) and their wild-type littermates (EC+/+) were injected with saline or lipopolysaccharide (18 mg/kg) to model sepsis-induced lung injury. At 18 h post-injection mice, were euthanized and bronchoalveolar lavage (BAL) fluid and lung tissue were collected to assess leucocyte infiltration. Poldip2 EC-/- mice showed reduced lung leucocyte infiltration in BAL (0.21 ± 0.9×106 vs. 1.29 ± 1.8×106 cells/mL) and lung tissue (12.7 ± 1.8 vs. 23 ± 3.7% neutrophils of total number of cells) compared to Poldip2 EC+/+ mice. qPCR analysis of the lung tissue revealed a significantly dampened induction of inflammatory gene expression (TNFα 2.23 ± 0.39 vs. 4.15 ± 0.5-fold, IκBα 4.32 ± 1.53 vs. 8.97 ± 1.59-fold), neutrophil chemoattractant gene expression (CXCL1 68.8 ± 29.6 vs. 147 ± 25.7-fold, CXCL2 65 ± 25.6 vs. 215 ± 27.3-fold) and a marker of endothelial activation (VCAM1 1.25 ± 0.25 vs. 3.8 ± 0.38-fold) in Poldip2 EC-/- compared to Poldip2 EC+/+ lungs. An in vitro model using human pulmonary microvascular endothelial cells was used to assess the effect of Poldip2 knock-down on endothelial activation and permeability. TNFα-induced endothelial permeability and VE-cadherin disruption were significantly reduced with siRNA-mediated knock-down of Poldip2 (5 ± 0.5 vs. 17.5 ± 3-fold for permeability, 1.5 ± 0.4 vs. 10.9 ± 1.3-fold for proportion of disrupted VE-cadherin). Poldip2 knock-down altered expression of Rho-GTPase-related genes, which correlated with reduced RhoA activation by TNFα (0.94 ± 0.05 vs. 1.29 ± 0.01 of relative RhoA activity) accompanied by redistribution of active-RhoA staining to the centre of the cell. CONCLUSION: Poldip2 is a potent regulator of endothelial dysfunction during sepsis-induced lung injury, and its endothelium-specific inhibition may provide clinical benefit.

Catalase overexpression in aortic smooth muscle prevents pathological mechanical changes underlying abdominal aortic aneurysm formation.

The causality of the associations between cellular and mechanical mechanisms of abdominal aortic aneurysm (AAA) formation has not been completely defined. Because reactive oxygen species are established mediators of AAA growth and remodeling, our objective was to investigate oxidative stress-induced alterations in aortic biomechanics and microstructure during subclinical AAA development. We investigated the mechanisms of AAA in an angiotensin II (ANG II) infusion model of AAA in apolipoprotein E-deficient (apoE(-/-)) mice that overexpress catalase in vascular smooth muscle cells (apoE(-/-)xTg(SMC-Cat)). At baseline, aortas from apoE(-/-)xTg(SMC-Cat) exhibited increased stiffness and the microstructure was characterized by 50% more collagen content and less elastin fragmentation. ANG II treatment for 7 days in apoE(-/-) mice altered the transmural distribution of suprarenal aortic circumferential strain (quantified by opening angle, which increased from 130 ± 1° at baseline to 198 ± 8° after 7 days of ANG II treatment) without obvious changes in the aortic microstructure. No differences in aortic mechanical behavior or suprarenal opening angle were observed in apoE(-/-)xTg(SMC-Cat) after 7 days of ANG II treatment. These data suggest that at the earliest stages of AAA development H(2)O(2) is functionally important and is involved in the control of local variations in remodeling across the vessel wall. They further suggest that reduced elastin integrity at baseline may predispose the abdominal aorta to aneurysmal mechanical remodeling.