Daiana Weiss

Publications of Daiana Weiss

• Reactive Oxygen Species Regulate Osteopontin Expression in a Murine Model of Postischemic Neovascularization.

  Authors: Alicia N Lyle, Giji Joseph, Aaron E Fan, Daiana Weiss, Natalia Landázuri, W Robert Taylor

  Arteriosclerosis, thrombosis, and vascular biology. 04/2012;

  OBJECTIVE: Previous findings from our laboratory demonstrated that neovascularization was impaired in osteopontin (OPN) knockout animals. However, the mechanisms responsible for the regulation of OPNOBJECTIVE: 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.

  Authors: Sarah F Knight, Kousik Kundu, Giji Joseph, Sergey Dikalov, Daiana Weiss, Niren Murthy, W Robert Taylor

  Journal of the American Society of Nephrology : JASN. 01/2012;

  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,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.

• Pharmacological suppression of hepcidin increases macrophage cholesterol efflux and reduces foam cell formation and atherosclerosis.

  Authors: Omar Saeed, Fumiyuki Otsuka, Rohini Polavarapu, Vinit Karmali, Daiana Weiss, Talina Davis, Brad Rostad, Kimberly Pachura, Lila Adams, John Elliott, W Robert Taylor, Jagat Narula, Frank Kolodgie, Renu Virmani, Charles C Hong, Aloke V Finn

  Arteriosclerosis, thrombosis, and vascular biology. 11/2011; 32(2):299-307.

  We recently reported that lowering of macrophage free intracellular iron increases expression of cholesterol efflux transporters ABCA1 and ABCG1 by reducing generation of reactive oxygen species. InWe recently reported that lowering of macrophage free intracellular iron increases expression of cholesterol efflux transporters ABCA1 and ABCG1 by reducing generation of reactive oxygen species. In this study, we explored whether reducing macrophage intracellular iron levels via pharmacological suppression of hepcidin can increase macrophage-specific expression of cholesterol efflux transporters and reduce atherosclerosis. To suppress hepcidin, increase expression of the iron exporter ferroportin, and reduce macrophage intracellular iron, we used a small molecule inhibitor of bone morphogenetic protein (BMP) signaling, LDN 193189 (LDN). LDN (10 mg/kg IP b.i.d.) was administered to mice, and its effects on atherosclerosis, intracellular iron, oxidative stress, lipid efflux, and foam cell formation were measured in plaques and peritoneal macrophages. Long-term LDN administration to apolipoprotein E-/- mice increased ABCA1 immunoreactivity within intraplaque macrophages by 3.7-fold (n=8; P=0.03), reduced Oil Red O-positive lipid area by 50% (n=8; P=0.02), and decreased total plaque area by 43% (n=8; P=0.001). LDN suppressed liver hepcidin transcription and increased macrophage ferroportin, lowering intracellular iron and hydrogen peroxide production. LDN treatment increased macrophage ABCA1 and ABCG1 expression, significantly raised cholesterol efflux to ApoA-1, and decreased foam cell formation. All preceding LDN-induced effects on cholesterol efflux were reversed by exogenous hepcidin administration, suggesting modulation of intracellular iron levels within macrophages as the mechanism by which LDN triggers these effects. These data suggest that pharmacological manipulation of iron homeostasis may be a promising target to increase macrophage reverse cholesterol transport and limit atherosclerosis.

• Overexpression of catalase in myeloid cells causes impaired postischemic neovascularization.

  Authors: Roberto Hodara, Daiana Weiss, Giji Joseph, Juan C Velasquez-Castano, Natalia Landázuri, Ji Woong Han, Young-sup Yoon, W Robert Taylor

  Arteriosclerosis, thrombosis, and vascular biology. 07/2011; 31(10):2203-9.

  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 theirMyeloid 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. 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. 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.

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

  Authors: Kathryn Maiellaro-Rafferty, Daiana Weiss, Giji Joseph, William Wan, Rudolph L Gleason, W Robert Taylor

  American journal of physiology. Heart and circulatory physiology. 05/2011; 301(2):H355-62.

  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 areThe 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.

• Effect of poly(ethylene glycol) diacrylate concentration on network properties and in vivo response of poly(β-amino ester) networks.

  Authors: David L Safranski, Daiana Weiss, J Brian Clark, Birgitta S Caspersen, W Robert Taylor, Ken Gall

  Journal of biomedical materials research. Part A. 02/2011; 96(2):320-9.

  Poly(β-amino ester) networks have shown promise as tissue scaffolds. The objective of this work was to examine the effect of changing poly(ethylene glycol) diacrylate concentration on poly(β-aminoPoly(β-amino ester) networks have shown promise as tissue scaffolds. The objective of this work was to examine the effect of changing poly(ethylene glycol) diacrylate concentration on poly(β-amino ester) network properties and to assess the degradable polymers' in vivo response, using magnetic resonance imaging (MRI) and immunohistochemistry. The networks were synthesized from hexanediol diacrylate (HDDA), poly(ethylene glycol) diacrylate (PEGDA), and a primary amine, 3-methoxypropylamine (3-MOPA), with a fixed overall molar ratio of diacrylate to amine. Network properties were verified to insure that the networks possessed equivalent initial properties and structure other than chemistry. The effect of varying PEGDA concentration on water content, mass loss, and modulus was determined, where increasing the concentration of PEGDA increases both water content, mass loss rate, and decreases modulus. We also show that manipulating the network composition at ratios of 0:100, 10:90 and 25:75 (PEGDA:HDDA) does not elicit a major inflammatory response to subcutaneous implantation of the networks in mice. This work provides a foundation for tailoring poly(β-amino ester) networks, based on degradation rate and modulus, as a means to tune the polymer properties for various biomedical applications.

• Preferential activation of SMAD1/5/8 on the fibrosa endothelium in calcified human aortic valves--association with low BMP antagonists and SMAD6.

  Authors: Randall F Ankeny, Vinod H Thourani, Daiana Weiss, J David Vega, W Robert Taylor, Robert M Nerem, Hanjoong Jo

  PloS one. 01/2011; 6(6):e20969.

  Aortic valve (AV) calcification preferentially occurs on the fibrosa side while the ventricularis side remains relatively unaffected. Here, we tested the hypothesis that side-dependent activation ofAortic valve (AV) calcification preferentially occurs on the fibrosa side while the ventricularis side remains relatively unaffected. Here, we tested the hypothesis that side-dependent activation of bone morphogenic protein (BMP) pathway in the endothelium of the ventricularis and fibrosa is associated with human AV calcification. Human calcified AVs obtained from AV replacement surgeries and non-calcified AVs from heart transplantations were used for immunohistochemical studies. We found SMAD-1/5/8 phosphorylation (a canonical BMP pathway) was higher in the calcified fibrosa than the non-calcified fibrosa while SMAD-2/3 phosphorylation (a canonical TGFβ pathway) did not show any difference. Interestingly, we found that BMP-2/4/6 expression was significantly higher on the ventricularis endothelium compared to the fibrosa in both calcified and non-calcified AV cusps; however, BMP antagonists (crossvienless-2/BMPER and noggin) expression was significantly higher on the ventricularis endothelium compared to the fibrosa in both disease states. Moreover, significant expression of inhibitory SMAD-6 expression was found only in the non-calcified ventricularis endothelium. SMAD-1/5/8 is preferentially activated in the calcified fibrosa endothelium of human AVs and it correlates with low expression of BMP antagonists and inhibitory SMAD6. These results suggest a dominant role of BMP antagonists in the side-dependent calcification of human AVs.

• Inhibition and genetic ablation of the B7/CD28 T-cell costimulation axis prevents experimental hypertension.

  Authors: Antony Vinh, Wei Chen, Yelena Blinder, Daiana Weiss, W Robert Taylor, Jörg J Goronzy, Cornelia M Weyand, David G Harrison, Tomasz J Guzik

  Circulation. 12/2010; 122(24):2529-37.

  The pathogenesis of hypertension remains poorly understood, and treatment is often unsuccessful. Recent evidence suggests that the adaptive immune response plays an important role in this disease.The pathogenesis of hypertension remains poorly understood, and treatment is often unsuccessful. Recent evidence suggests that the adaptive immune response plays an important role in this disease. Various hypertensive stimuli cause T-cell activation and infiltration into target organs such as the vessel and the kidney, which promotes vascular dysfunction and blood pressure elevation. Classically, T-cell activation requires T-cell receptor ligation and costimulation. The latter often involves interaction between B7 ligands (CD80 and CD86) on antigen-presenting cells with the T-cell coreceptor CD28. This study was therefore performed to examine the role of this pathway in hypertension. Angiotensin II-induced hypertension increased the presence of activated (CD86(+)) dendritic cells in secondary lymphatic tissues. Blockade of B7-dependent costimulation with CTLA4-Ig reduced both angiotensin II- and deoxycorticosterone acetate (DOCA)-salt-induced hypertension. Activation of circulating T cells, T-cell cytokine production, and vascular T-cell accumulation caused by these hypertensive stimuli were abrogated by CTLA4-Ig. Furthermore, in mice lacking B7 ligands, angiotensin II caused minimal blood pressure elevation and vascular inflammation, and these effects were restored by transplantation with wild-type bone marrow. T-cell costimulation via B7 ligands is essential for development of experimental hypertension, and inhibition of this process could have therapeutic benefit in the treatment of this disease.

• Sustained VEGF delivery via PLGA nanoparticles promotes vascular growth.

  Authors: Justin S Golub, Young-tae Kim, Craig L Duvall, Ravi V Bellamkonda, Divya Gupta, Angela S Lin, Daiana Weiss, W Robert Taylor, Robert E Guldberg

  American journal of physiology. Heart and circulatory physiology. 03/2010; 298(6):H1959-65.

  Technologies to increase tissue vascularity are critically important to the fields of tissue engineering and cardiovascular medicine. Currently, limited technologies exist to encourage angiogenesisTechnologies to increase tissue vascularity are critically important to the fields of tissue engineering and cardiovascular medicine. Currently, limited technologies exist to encourage angiogenesis and arteriogenesis in a controlled manner. In the present study, we describe an injectable controlled release system consisting of VEGF encapsulated in poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs). The majority of VEGF was released gradually over 2-4 days from the NPs as determined by an ELISA release kinetics experiment. An in vitro aortic ring bioassay was used to verify the bioactivity of VEGF-NPs compared with empty NPs and no treatment. A mouse femoral artery ischemia model was then used to measure revascularization in VEGF-NP-treated limbs compared with limbs treated with naked VEGF and saline. 129/Sv mice were anesthetized with isoflurane, and a region of the common femoral artery and vein was ligated and excised. Mice were then injected with VEGF-NPs, naked VEGF, or saline. After 4 days, three-dimensional microcomputed tomography angiography was used to quantify vessel growth and morphology. Mice that received VEGF-NP treatment showed a significant increase in total vessel volume and vessel connectivity compared with 5 microg VEGF, 2.5 microg VEGF, and saline treatment (all P < 0.001). When the yield of the fabrication process was taken into account, VEGF-NPs were over an order of magnitude more potent than naked VEGF in increasing blood vessel volume. Differences between the VEGF-NP group and all other groups were even greater when only small-sized vessels under 300 mum diameter were analyzed. In conclusion, sustained VEGF delivery via PLGA NPs shows promise for encouraging blood vessel growth in tissue engineering and cardiovascular medicine applications.

• Vascular wall ACE is not required for atherogenesis in ApoE(-/-) mice.

  Authors: Daiana Weiss, Kenneth E Bernstein, Sebastian Fuchs, Jonathan Adams, Andreas Synetos, W Robert Taylor

  Atherosclerosis. 10/2009;

  BACKGROUND: It has been proposed that elements of the renin angiotensin system expressed in the arterial wall are critical for the development of atherosclerosis. Angiotensin converting enzyme (ACE)BACKGROUND: It has been proposed that elements of the renin angiotensin system expressed in the arterial wall are critical for the development of atherosclerosis. Angiotensin converting enzyme (ACE) is highly expressed by the endothelium and is responsible for a critical enzymatic step in the generation of angiotensin II. However, the functional contribution of ACE expression in the vascular wall in atherogenesis is unknown. Therefore, we made use of unique genetic models in which mice without the expression of ACE in the vascular wall were crossed with ApoE(-/-) mice in order to determine the contribution of tissue ACE expression to atherosclerotic lesion formation. METHODS AND RESULTS: Mice expressing either a soluble form of ACE (ACE 2/2) or mice with somatic ACE expression restricted to the liver and kidney (ACE 3/3) on an ApoE(-/-) background were placed on a standard chow or Western diet for 6 months. Atherosclerotic lesion area in the ACE 2/2 mice was significantly lower than that seen in the ACE 3/3 mice. However, these animals also had significantly lower blood pressure and reduced plasma ACE activity which precluded establishing a specific causal relationship between absent tissue ACE activity and decreased atherosclerotic lesion extent. Therefore, we studied the ACE 3/3 mice which are normotensive and lack vascular ACE expression. In the ACE 3/3 animals, atherosclerotic lesion area was not different from wild type controls despite reduced plasma ACE activity. CONCLUSIONS: We concluded that under these experimental conditions, expression of ACE in the arterial wall is not required for atherosclerotic lesion formation.

• Expression of CYP1A1 and CYP1B1 in human endothelial cells: Regulation by fluid shear stress.

  Authors: Daniel E Conway, Yumiko Sakurai, Daiana Weiss, J David Vega, W Robert Taylor, Hanjoong Jo, Suzanne G Eskin, Craig B Marcus, Larry V McIntire

  Cardiovascular research. 02/2009;

  AIM: CYP1A1 and CYP1B1, members of the cytochrome P450 protein family, are regulated by fluid shear stress. This study describes the effects of duration, magnitude and pattern of shear stress onAIM: CYP1A1 and CYP1B1, members of the cytochrome P450 protein family, are regulated by fluid shear stress. This study describes the effects of duration, magnitude and pattern of shear stress on CYP1A1 and CYP1B1 expression in human endothelial cells, toward the goal of understanding the role(s) of these genes in pro-atherogenic or anti-atherogenic endothelial cell functions. Methods and Results We investigated CYP1A1 and CYP1B1 expression under different durations, levels, and patterns of shear stress. CYP1A1 and CYP1B1 mRNA, protein, and enzymatic activity were maximally up-regulated at equal to or greater than 24 hours of arterial levels of shear stress (15-25 dynes/cm(2)). Expression of both genes was significantly attenuated by reversing shear stress as compared to 15 dynes/cm(2) steady shear stress. Small interfering RNA knockdown of CYP1A1 resulted in significantly reduced CYP1B1 and thrombospondin-1 expression, genes regulated by the aryl hydrocarbon receptor (AhR). Immunostaining of human coronary arteries showed constitutive CYP1A1 and CYP1B1 protein expression in endothelial cells. Immunostaining of mouse aorta showed nuclear localization of AhR and increased expression of CYP1A1 in the descending thoracic aorta, whereas reduced nuclear localization of AhR and attenuated CYP1A1 expression were observed in the lesser curvature of the aortic arch. CONCLUSIONS: CYP1A1 and CYP1B1 gene and protein expressions vary with time, magnitude, and pattern of shear stress. Increased CYP1A1 gene expression modulates AhR regulated genes. Based on our in vitro reversing flow data and in vivo immunostained mouse aorta, we suggest that increased expression of both genes reflects an anti-atherogenic endothelial cell phenotype.

• The role of osteopontin in recovery from hind limb ischemia.

  Authors: Craig L Duvall, Daiana Weiss, Scott T Robinson, Fadi M F Alameddine, Robert E Guldberg, W Robert Taylor

  Arteriosclerosis, thrombosis, and vascular biology. 03/2008; 28(2):290-5.

  OBJECTIVE: Osteopontin (OPN) is a highly phosphorylated extracellular matrix glycoprotein that is involved in a diversity of biological processes. In the vascular wall, OPN is produced byOBJECTIVE: Osteopontin (OPN) is a highly phosphorylated extracellular matrix glycoprotein that is involved in a diversity of biological processes. In the vascular wall, OPN is produced by monocytes/macrophages, endothelial cells, and smooth muscle cells, and it is thought to mediate adhesion, migration, and survival of these cell types. In this study, we hypothesized that OPN plays a critical role in recovery from limb ischemia. METHODS AND RESULTS: We induced hind limb ischemia in wild-type and OPN-/- mice. OPN-/- mice exhibited significantly delayed recovery of ischemic foot perfusion as determined by LDPI, impaired collateral vessel formation as measured using micro-CT, and diminished functional capacity of the ischemic limb. In the aortic ring assay, normal endothelial cell sprouting was found in OPN-/- mice. However, OPN-/- peritoneal monocytes/macrophages were found to possess significantly reduced migration in response to chemoattraction. CONCLUSIONS: This study provides evidence that a definitive biological role exists for OPN during ischemic limb revascularization, and we have suggested that this may be driven by impaired monocyte/macrophage migration in OPN-/- mice. These findings provide the first in vivo evidence that OPN may be a key regulator in postnatal vascular growth.

• Deoxycorticosterone acetate salt hypertension in apolipoprotein E-/- mice results in accelerated atherosclerosis: the role of angiotensin II.

  Authors: Daiana Weiss, W Robert Taylor

  Hypertension. 03/2008; 51(2):218-24.

  Previous studies have shown that administration of angiotensin II to atherosclerosis-prone animal models results in an increase in the extent of atherosclerosis and that this effect may bePrevious studies have shown that administration of angiotensin II to atherosclerosis-prone animal models results in an increase in the extent of atherosclerosis and that this effect may be independent of changes in blood pressure. We sought to determine whether atherosclerosis was increased in the setting of a low renin model of hypertension. Apolipoprotein E-deficient mice were made hypertensive using the deoxycorticosterone acetate salt model. We found that this resulted in a dramatic increase in the atherosclerotic lesion area in the setting of either a low- or high-fat diet. In the hypertensive animals, we observed an increase in angiotensin II staining that was localized to the adventitial macrophages. The increase in atherosclerosis was inhibited by administration of an angiotensin receptor antagonist, an angiotensin-converting enzyme inhibitor, or a renin inhibitor. In addition, blood pressure reduction, with either a calcium channel blocker or hydralazine, reduced the extent of atherosclerosis indicating an important contribution of the mechanical effects of elevated blood pressure. These data suggest that, even in the setting of hypertension that is not associated with activation of the systemic renin-angiotensin system, local generation of angiotensin II within the arterial wall may be of pathophysiological relevance to the development of atherosclerosis.

• Differential effects of AT1 receptor and Ca2+ channel blockade on atherosclerosis, inflammatory gene expression, and production of reactive oxygen species.

  Authors: Derek E Doran, Daiana Weiss, Yong Zhang, Kathy K Griendling, W Robert Taylor

  Atherosclerosis. 12/2007; 195(1):39-47.

  Angiotensin II receptor blockade has been shown to inhibit atherosclerosis in several different animal models. We sought to determine if this effect was the result of blood pressure reduction per seAngiotensin II receptor blockade has been shown to inhibit atherosclerosis in several different animal models. We sought to determine if this effect was the result of blood pressure reduction per se or a result of the anti-inflammatory effects of receptor blockade. ApoE-deficient mice were fed a high fat diet and treated with either an angiotensin II receptor antagonist, candesartan (0.5 mg/kg/day, s.c.) or a calcium channel blocker, amlodipine (7.5 mg/kg/day, mixed with food). Atherosclerotic lesion area, aortic inflammatory gene expression as well as aortic H2O2 and superoxide production were assayed. We found that candesartan but not amlodipine treatment dramatically attenuated the development of atherosclerosis despite a similar reduction in blood pressure. Similarly, candesartan treatment inhibited aortic expression of inflammatory genes and production of reactive oxygen species, effects not seen with amlodipine. These data demonstrate that angiotensin II receptor blockade inhibits atherosclerosis by reducing vascular oxidative stress and inflammatory gene production independent of blood pressure reduction.

• Human heart failure is associated with abnormal C-terminal splicing variants in the cardiac sodium channel.

  Authors: Lijuan L Shang, Arnold E Pfahnl, Shamarendra Sanyal, Zhe Jiao, Jon Allen, Kathrin Banach, John Fahrenbach, Daiana Weiss, W Robert Taylor, A Maziar Zafari, Samuel C Dudley

  Circulation research. 12/2007; 101(11):1146-54.

  Heart failure (HF) is associated with reduced cardiac Na+ channel (SCN5A) current. We hypothesized that abnormal transcriptional regulation of this ion channel during HF could help explain theHeart failure (HF) is associated with reduced cardiac Na+ channel (SCN5A) current. We hypothesized that abnormal transcriptional regulation of this ion channel during HF could help explain the reduced current. Using human hearts explanted at the transplantation, we have identified 3 human C-terminal SCN5A mRNA splicing variants predicted to result in truncated, nonfunctional channels. As compared with normal hearts, the explanted ventricles showed an upregulation of 2 of the variants and a downregulation of the full-length mRNA transcript such that the E28A transcript represented only 48.5% (P<0.01) of the total SCN5A mRNA. This correlated with a 62.8% (P<0.01) reduction in Na+ channel protein. Lymphoblasts and skeletal muscle expressing SCN5A also showed identical C-terminal splicing variants. Variants showed reduced membrane protein and no functional current. Transfection of truncation variants into a cell line stably transfected with the full-length Na+ channel resulted in dose-dependent reductions in channel mRNA and current. Introduction of a premature truncation in the C-terminal region in a single allele of the mouse SCN5A resulted in embryonic lethality. Embryonic stem cell-derived cardiomyocytes expressing the construct showed reductions in Na+ channel-dependent electrophysiological parameters, suggesting that the presence of truncated Na+ channel mRNA at levels seen in HF is likely to be physiologically significant. In summary, chronic HF was associated with an increase in 2 truncated SCN5A variants and a decrease in the native mRNA. These splice variations may help explain a loss of Na+ channel protein and may contribute to the increased arrhythmic risk in clinical HF.

• Bone morphogenic protein antagonists are coexpressed with bone morphogenic protein 4 in endothelial cells exposed to unstable flow in vitro in mouse aortas and in human coronary arteries: role of bone morphogenic protein antagonists in inflammation and atherosclerosis.

  Authors: Kyunghwa Chang, Daiana Weiss, Jin Suo, J David Vega, Don Giddens, W Robert Taylor, Hanjoong Jo

  Circulation. 10/2007; 116(11):1258-66.

  BACKGROUND: Exposure to disturbed flow, including oscillatory shear stress, stimulates endothelial cells (ECs) to produce bone morphogenic protein (BMP) 4, which in turn activates inflammation, aBACKGROUND: Exposure to disturbed flow, including oscillatory shear stress, stimulates endothelial cells (ECs) to produce bone morphogenic protein (BMP) 4, which in turn activates inflammation, a critical atherogenic step. BMP activity is regulated by the level of BMP antagonists. Until now it was not known whether shear also regulates the expression of BMP antagonists and whether they play a role in EC pathophysiology. METHODS AND RESULTS: BMP antagonists follistatin, noggin, and matrix Gla protein were expressed in cultured bovine and human arterial ECs. Surprisingly, oscillatory shear stress increased expression of the BMP antagonists in ECs, whereas unidirectional laminar shear decreased such expression. Immunohistochemical studies with mouse aortas showed data consistent with in vitro findings: Only ECs in the lesser curvature exposed to disturbed flow, but not those in the greater curvature and straight arterial regions exposed to undisturbed flow, showed coexpression of BMP4 and the BMP antagonists. Similarly, in human coronary arteries, expression of BMP4 and BMP antagonists in ECs positively correlated with the severity of atherosclerosis. Monocyte adhesion induced by oscillatory shear stress was inhibited by knockdown of BMP4 or treatment with recombinant follistatin or noggin, whereas it was increased by knockdown of follistatin and/or noggin. CONCLUSIONS: The present results suggest that ECs coexpress BMP antagonists along with BMP4 in an attempt to minimize the inflammatory response by oscillatory shear stress as part of a negative feedback mechanism. The balance between the agonist, BMP4, and its antagonists may play an important role in the overall control of inflammation and atherosclerosis.

• Mice with enhanced macrophage angiotensin-converting enzyme are resistant to melanoma.

  Authors: Xiao Z Shen, Ping Li, Daiana Weiss, Sebastien Fuchs, Hong D Xiao, Jon A Adams, Ifor R Williams, Mario R. Capecchi, W Robert Taylor, Kenneth E Bernstein

  The American journal of pathology. 07/2007; 170(6):2122-34.

  Angiotensin-converting enzyme (ACE) is a peptidase responsible for the cleavage of angiotensin I and several other peptides. Here, gene targeting was used to switch control of the ACE locus from theAngiotensin-converting enzyme (ACE) is a peptidase responsible for the cleavage of angiotensin I and several other peptides. Here, gene targeting was used to switch control of the ACE locus from the endogenous promoter to the macrophage-specific c-fms promoter. Challenge of these mice, called ACE 10/10, with the aggressive mouse melanoma cell line B16 showed that they are remarkably resistant to tumor growth. Tumor resistance was seen after challenge with different melanoma cell lines and in mice with different genetic backgrounds. Histological study of the tumors that did grow in ACE 10/10 mice showed an enhanced inflammatory response. ACE 10/10 mice had increased numbers of tumor epitope-specific CD8(+) T cells after challenge with melanoma or lymphoma. ACE 10/10 macrophages showed increased production of interleukin-12 and nitric oxide but reduced interleukin-10. Engraftment of wild-type mice with ACE 10/10 bone marrow transferred B16 tumor resistance. Injection of B16 tumors with ACE 10/10 macrophages also reduced tumor growth. ACE 10/10 mice may define a new means of enhancing the immune response, which may be potentially useful in several human clinical situations.

• Granulocyte colony-stimulating factor and granulocyte macrophage colony-stimulating factor exacerbate atherosclerosis in apolipoprotein E-deficient mice.

  Authors: Amir Haghighat, Daiana Weiss, Matthew K Whalin, D Patrick Cowan, W Robert Taylor

  Circulation. 04/2007; 115(15):2049-54.

  Recent studies have suggested a potential contribution of bone marrow-derived progenitor cells to vascular repair. Preliminary clinical studies have explored the possibility that mobilization ofRecent studies have suggested a potential contribution of bone marrow-derived progenitor cells to vascular repair. Preliminary clinical studies have explored the possibility that mobilization of progenitor cells with granulocyte macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) can affect vascular repair. However, it is not known whether the short-term administration of G-CSF or GM-CSF exerts beneficial effects on atherosclerosis. Apolipoprotein E-deficient mice were treated with either GM-CSF or G-CSF at a dose of 10 microg x kg(-1) x d(-1) s.c. administered daily for 5 days per week on alternating weeks for a total of 20 doses over an 8-week treatment period. We found that in animals maintained on a high-fat diet, both G-CSF and GM-CSF actually demonstrated an increase in atherosclerotic lesion extent. The increase in atherosclerotic extent was not associated with an increase in either inflammatory cells or expression of proinflammatory genes. Interestingly, adventitial vascularity significantly increased, suggesting a mechanistic role for vasa vasorum neovascularization. These findings demonstrate that in this animal model of atherosclerosis, not only did administration of G-CSF or GM-CSF fail to demonstrate any beneficial therapeutic effect, but both resulted in a worsening of atherosclerosis.

• Expression of cathepsin K is regulated by shear stress in cultured endothelial cells and is increased in endothelium in human atherosclerosis.

  Authors: Manu O Platt, Randall F Ankeny, Guo-Ping Shi, Daiana Weiss, J D Vega, W R Taylor, Hanjoong Jo

  American journal of physiology. Heart and circulatory physiology. 04/2007; 292(3):H1479-86.

  Cathepsins, the lysosomal cysteine proteases, are involved in vascular remodeling and atherosclerosis. Genetic knockout of cathepsins S and K in mice has shown to reduce atherosclerosis, although theCathepsins, the lysosomal cysteine proteases, are involved in vascular remodeling and atherosclerosis. Genetic knockout of cathepsins S and K in mice has shown to reduce atherosclerosis, although the molecular mechanisms remain unclear. Because atherosclerosis preferentially occurs in arteries exposed to disturbed flow conditions, we hypothesized that shear stress would regulate cathepsin K expression and activity in endothelial cells. Mouse aortic endothelial cells (MAEC) exposed to proatherogenic oscillatory shear (OS, +/- 5 dyn/cm(2) for 1 day) showed significantly higher cathepsin K expression and activity than that of atheroprotective, unidirectional laminar shear stress (LS, 15 dyn/cm(2) for 1 day). Western blot and active-site labeling studies showed an active, mature form of cathepsin K in the conditioned medium of MAEC exposed to OS but not in that of LS. Functionally, MAEC exposed to OS significantly increased elastase and gelatinase activity above that of LS. The OS-dependent elastase and gelatinase activities were significantly reduced by knocking down cathepsin K with small-interfering (si) RNA, but not by a nonsilencing siRNA control, suggesting that cathepsin K is a shear-sensitive protease. In addition, immunohistochemical analysis of atherosclerotic human coronary arteries showed a positive correlation between the cathepsin K expression levels in endothelium and elastic lamina integrity. These findings suggest that cathepsin K is a mechanosensitive, extracellular matrix protease that, in turn, may be involved in arterial wall remodeling and atherosclerosis.

• Impaired angiogenesis, early callus formation, and late stage remodeling in fracture healing of osteopontin-deficient mice.

  Authors: Craig L Duvall, W Robert Taylor, Daiana Weiss, Abigail M Wojtowicz, Robert E Guldberg

  Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 03/2007; 22(2):286-97.

  OPN is an ECM protein with diverse localization and functionality. The role of OPN during fracture healing was examined using wildtype and OPN(-/-) mice. Results showed that OPN plays an importantOPN is an ECM protein with diverse localization and functionality. The role of OPN during fracture healing was examined using wildtype and OPN(-/-) mice. Results showed that OPN plays an important role in regulation of angiogenesis, callus formation, and mechanical strength in early stages of healing and facilitates late stage bone remodeling and ECM organization. INTRODUCTION: Osteopontin (OPN) is an extracellular matrix (ECM) protein with diverse localization and functionality that has been reported to play a regulatory role in both angiogenesis and osteoclastic bone remodeling, two vital processes for normal bone healing. MATERIALS AND METHODS: Bone repair in wildtype and OPN(-/-) mice was studied using a femoral fracture model. microCT was used for quantitative angiographic measurements at 7 and 14 days and to assess callus size and mineralization at 7, 14, 28, and 56 days. Biomechanical testing was performed on intact bones and on fracture specimens at 14, 28, and 56 days. Histology and quantitative RT-PCR were used to evaluate cellular functions related to ECM formation and bone remodeling. RESULTS: OPN deficiency was validated in the OPN(-/-) mice, which generally displayed normal levels of related ECM proteins. Intact OPN(-/-) bones displayed increased elastic modulus but decreased strength and ductility. Fracture neovascularization was reduced at 7 but not 14 days in OPN(-/-) mice. OPN(-/-) mice exhibited smaller fracture calluses at 7 and 14 days, as well as lower maximum torque and work to failure. At 28 days, OPN(-/-) mice had normal callus size but a persistent reduction in maximum torque and work to failure. Osteoclast differentiation occurred normally, but mature osteoclasts displayed reduced functionality, decreasing late stage remodeling in OPN(-/-) mice. Thus, at 56 days, OPN(-/-) fractures possessed increased callus volume, increased mechanical stiffness, and altered collagen fiber organization. CONCLUSIONS: This study showed multiple, stage-dependent roles of OPN during fracture healing. We conclude that OPN deficiency alters the functionality of multiple cell types, resulting in delayed early vascularization, altered matrix organization and late remodeling, and reduced biomechanical properties. These findings contribute to an improved understanding of the role of OPN in vivo and provide new insight into mechanistic control of vascularization and bone regeneration during fracture repair.