Multimodality Imaging of Aortic Valve Calcification and Function in a Murine Model of Calcific Aortic Valve Disease and Bicuspid Aortic Valve.

Calcific aortic valve disease (CAVD) is a prevailing disease with increasing occurrence and no known medical therapy. Dcbld2-/- mice have a high prevalence of bicuspid aortic valve (BAV), spontaneous aortic valve calcification, and aortic stenosis (AS). 18F-NaF PET/CT can detect the aortic valve calcification process in humans. However, its feasibility in preclinical models of CAVD remains to be determined. Here, we sought to validate 18F-NaF PET/CT for tracking murine aortic valve calcification and leveraged it to examine the development of calcification with aging and its interdependence with BAV and AS in Dcbld2-/- mice. Methods: Dcbld2-/- mice at 3-4 mo, 10-16 mo, and 18-24 mo underwent echocardiography, 18F-NaF PET/CT (n = 34, or autoradiography (n = 45)), and tissue analysis. A subset of mice underwent both PET/CT and autoradiography (n = 12). The aortic valve signal was quantified as SUVmax on PET/CT and as percentage injected dose per square centimeter on autoradiography. The valve tissue sections were analyzed by microscopy to identify tricuspid and bicuspid aortic valves. Results: The aortic valve 18F-NaF signal on PET/CT was significantly higher at 18-24 mo (P < 0.0001) and 10-16 mo (P < 0.05) than at 3-4 mo. Additionally, at 18-24 mo BAV had a higher 18F-NaF signal than tricuspid aortic valves (P < 0.05). These findings were confirmed by autoradiography, with BAV having significantly higher 18F-NaF uptake in each age group. A significant correlation between PET and autoradiography data (Pearson r = 0.79, P < 0.01) established the accuracy of PET quantification. The rate of calcification with aging was significantly faster for BAV (P < 0.05). Transaortic valve flow velocity was significantly higher in animals with BAV at all ages. Finally, there was a significant correlation between transaortic valve flow velocity and aortic valve calcification by both PET/CT (r = 0.55, P < 0.001) and autoradiography (r = 0.45, P < 0.01). Conclusion: 18F-NaF PET/CT links valvular calcification to BAV and aging in Dcbld2-/- mice and suggests that AS may promote calcification. In addition to addressing the pathobiology of valvular calcification, 18F-NaF PET/CT may be a valuable tool for evaluation of emerging therapeutic interventions in CAVD.

Homeostatic, Non-Canonical Role of Macrophage Elastase in Vascular Integrity.

BACKGROUND: Matrix metalloproteinase (MMP)-12 is highly expressed in abdominal aortic aneurysms and its elastolytic function has been implicated in the pathogenesis. This concept is challenged, however, by conflicting data. Here, we sought to revisit the role of MMP-12 in abdominal aortic aneurysm. METHODS: Apoe-/- and Mmp12-/-/Apoe-/- mice were infused with Ang II (angiotensin). Expression of neutrophil extracellular traps (NETs) markers and complement component 3 (C3) levels were evaluated by immunostaining in aortas of surviving animals. Plasma complement components were analyzed by immunoassay. The effects of a complement inhibitor, IgG-FH1-5 (factor H-immunoglobulin G), and macrophage-specific MMP-12 deficiency on adverse aortic remodeling and death from rupture in Ang II-infused mice were determined. RESULTS: Unexpectedly, death from aortic rupture was significantly higher in Mmp12-/-/Apoe-/- mice. This associated with more neutrophils, citrullinated histone H3 and neutrophil elastase, markers of NETs, and C3 levels in Mmp12-/- aortas. These findings were recapitulated in additional models of abdominal aortic aneurysm. MMP-12 deficiency also led to more pronounced elastic laminae degradation and reduced collagen integrity. Higher plasma C5a in Mmp12-/- mice pointed to complement overactivation. Treatment with IgG-FH1-5 decreased aortic wall NETosis and reduced adverse aortic remodeling and death from rupture in Ang II-infused Mmp12-/- mice. Finally, macrophage-specific MMP-12 deficiency recapitulated the effects of global MMP-12 deficiency on complement deposition and NETosis, as well as adverse aortic remodeling and death from rupture in Ang II-infused mice. CONCLUSIONS: An MMP-12 deficiency/complement activation/NETosis pathway compromises aortic integrity, which predisposes to adverse vascular remodeling and abdominal aortic aneurysm rupture. Considering these new findings, the role of macrophage MMP-12 in vascular homeostasis demands re-evaluation of MMP-12 function in diverse settings.

Differential BMP Signaling Mediates the Interplay Between Genetics and Leaflet Numbers in Aortic Valve Calcification.

Expression of a neuropilin-like protein, DCBLD2, is reduced in human calcific aortic valve disease (CAVD). DCBLD2-deficient mice develop bicuspid aortic valve (BAV) and CAVD, which is more severe in BAV mice compared with tricuspid littermates. In vivo and in vitro studies link this observation to up-regulated bone morphogenic protein (BMP)2 expression in the presence of DCBLD2 down-regulation, and enhanced BMP2 signaling in BAV, indicating that a combination of genetics and BAV promotes aortic valve calcification and stenosis. This pathway may be a therapeutic target to prevent CAVD progression in BAV.

Computed tomography imaging of macrophage phagocytic activity in abdominal aortic aneurysm.

Inflammation plays a major role in the pathogenesis of several vascular pathologies, including abdominal aortic aneurysm (AAA). Evaluating the role of inflammation in AAA pathobiology and potentially outcome in vivo requires non-invasive tools for high-resolution imaging. We investigated the feasibility of X-ray computed tomography (CT) imaging of phagocytic activity using nanoparticle contrast agents to predict AAA outcome. Methods: Uptake of several nanoparticle CT contrast agents was evaluated in a macrophage cell line. The most promising agent, Exitron nano 12000, was further characterized in vitro and used for subsequent in vivo testing. AAA was induced in Apoe-/- mice through angiotensin II (Ang II) infusion for up to 4 weeks. Nanoparticle biodistribution and uptake in AAA were evaluated by CT imaging in Ang II-infused Apoe-/- mice. After imaging, the aortic tissue was harvested and used from morphometry, transmission electron microscopy and gene expression analysis. A group of Ang II-infused Apoe-/- mice underwent nanoparticle-enhanced CT imaging within the first week of Ang II infusion, and their survival and aortic external diameter were evaluated at 4 weeks to address the value of vessel wall CT enhancement in predicting AAA outcome. Results: Exitron nano 12000 showed specific uptake in macrophages in vitro. Nanoparticle accumulation was observed by CT imaging in tissues rich in mononuclear phagocytes. Aortic wall enhancement was detectable on delayed CT images following nanoparticle administration and correlated with vessel wall CD68 expression. Transmission electron microscopy ascertained the presence of nanoparticles in AAA adventitial macrophages. Nanoparticle-induced CT enhancement on images obtained within one week of AAA induction was predictive of AAA outcome at 4 weeks. Conclusions: By establishing the feasibility of CT-based molecular imaging of phagocytic activity in AAA, this study links the inflammatory signal on early time point images to AAA evolution. This readily available technology overcomes an important barrier to cross-sectional, longitudinal and outcome studies, not only in AAA, but also in other cardiovascular pathologies and facilitates the evaluation of modulatory interventions, and ultimately upon clinical translation, patient management.

Hydroxamate-Based Selective Macrophage Elastase (MMP-12) Inhibitors and Radiotracers for Molecular Imaging.

Macrophage elastase [matrix metalloproteinase (MMP)-12] is the most upregulated MMP in abdominal aortic aneurysm (AAA) and, hence, MMP-12-targeted imaging may predict AAA progression and rupture risk. Here, we report the design, synthesis, and evaluation of three novel hydroxamate-based selective MMP-12 inhibitors (CGA, CGA-1, and AGA) and the methodology to obtain MMP-12 selectivity from hydroxamate-based panMMP inhibitors. Also, we report two 99mTc-radiotracers, 99mTc-AGA-1 and 99mTc-AGA-2, derived from AGA. 99mTc-AGA-2 displayed faster blood clearance in mice and better radiochemical stability compared to 99mTc-AGA-1. Based on this, 99mTc-AGA-2 was chosen as the lead tracer and tested in murine AAA. 99mTc-AGA-2 uptake detected by autoradiography was significantly higher in AAA compared to normal aortic regions. Specific binding of the tracer to MMP-12 was demonstrated through ex vivo competition. Accordingly, this study introduces a novel family of selective MMP-12 inhibitors and tracers, paving the way for further development of these agents as therapeutic and imaging agents.

Novel Matrix Metalloproteinase 12 Selective Radiotracers for Vascular Molecular Imaging.

Matrix metalloproteinase-12 (MMP-12) is highly upregulated in several inflammatory diseases, including abdominal aortic aneurysm (AAA). Here we report four novel 99mTc-labeled radiotracers derived from a highly selective competitive MMP-12 inhibitor. These tracers in their 99gTc version were assessed in vitro on a set of human metalloproteases and displayed high affinity and selectivity toward MMP-12. Their radiolabeling with 99mTc was shown to be efficient and stable in both buffer and mouse blood. The tracers showed major differences in their biodistribution and blood clearance. On the basis of its in vivo performance, [99mTc]-1 was selected for evaluation in murine AAA, where MMP-12 gene expression is upregulated. Autoradiography of aortae at 2 h postinjection revealed high uptake of [99mTc]-1 in AAA relative to adjacent aorta. Tracer uptake specificity was demonstrated through in vivo competition. This study paves the way for further evaluation of [99mTc]-1 for imaging AAA and other MMP-12-associated diseases.

Novel Arginine-containing Macrocyclic MMP Inhibitors: Synthesis, 99mTc-labeling, and Evaluation.

Matrix metalloproteinases (MMPs) are involved in tissue remodeling. Accordingly, MMP inhibitors and related radiolabeled analogs are important tools for MMP-targeted imaging and therapy in a number of diseases. Herein, we report design, synthesis, and evaluation of a new Arginine-containing macrocyclic hydroxamate analog, RYM, its hydrazinonicotinamide conjugate, RYM1 and 99mTc-labeled analog 99mTc-RYM1 for molecular imaging. RYM exhibited potent inhibition against a panel of recombinant human (rh) MMPs in vitro. RYM1 was efficiently labeled with 99mTcO4- to give 99mTc-RYM1 in a high radiochemical yield and high radiochemical purity. RYM1 and its decayed labeling product displayed similar inhibition potencies against rhMMP-12. Furthermore, 99mTc-RYM1 exhibited specific binding with lung tissue from lung-specific interleukin-13 transgenic mice, in which MMP activity is increased in conjunction with tissue remodeling and inflammation. The results support further development of such new water-soluble Arginine-containing macrocyclic hydroxamate MMP inhibitors for targeted imaging and therapy.

Molecular imaging of calcific aortic valve disease.

Calcific aortic valve disease (CAVD) can progress to symptomatic aortic stenosis in a subset of patients. The severity of aortic stenosis and the extent of valvular calcification can be evaluated readily by echocardiography, CT, and MRI using well-established imaging protocols. However, these techniques fail to address optimally other important aspects of CAVD, including the propensity for disease progression, risk of complications in asymptomatic patients, and the effect of therapeutic interventions on valvular biology. These gaps may be addressed by molecular imaging targeted at key biological processes such as inflammation, remodeling, and calcification that mediate the development and progression of CAVD. In this review, recent advances in valvular molecular imaging, including 18F-fluorodeoxyglucose (FDG) and 18F-sodium fluoride (NaF) PET, and matrix metalloproteinase-targeted SPECT imaging in the preclinical and clinical settings are presented and discussed.

Preclinical Evaluation of RYM1, a Matrix Metalloproteinase-Targeted Tracer for Imaging Aneurysm.

Matrix metalloproteinases (MMPs) play a key role in abdominal aortic aneurysm (AAA) development. Accordingly, MMP-targeted imaging provides important information regarding vessel wall biology in the course of aneurysm development. Given the small size of the vessel wall and its proximity with blood, molecular imaging of aneurysm optimally requires highly sensitive tracers with rapid blood clearance. To this end, we developed a novel hydrosoluble zwitterionic MMP inhibitor, RYM, on the basis of which a pan-MMP tracer, RYM1, was designed. Here, we describe the development and preclinical evaluation of RYM1 in comparison with RP805, a commonly used pan-MMP tracer in murine models of aneurysm. Methods: The macrocyclic hydroxamate-based pan-MMP inhibitor coupled with 6-hydrazinonicotinamide, RYM1, was synthesized and labeled with 99mTc. Radiochemical stability of 99mTc-RYM1 was evaluated by radio-high-performance liquid chromatography analysis. Tracer blood kinetics and biodistribution were compared with 99mTc-RP805 in C57BL/6J mice (n = 10). 99mTc-RYM1 binding to aneurysm and specificity were evaluated by quantitative autoradiography in apolipoprotein E-deficient (apoE-/-) mice with CaCl2-induced carotid aneurysm (n = 11). Angiotensin II-infused apoE-/- (n = 16) mice were used for small-animal SPECT/CT imaging. Aortic tissue MMP activity and macrophage marker CD68 expression were assessed by zymography and reverse-transcription polymerase chain reaction. Results: RYM1 showed nanomolar range inhibition constants for several MMPs. 99mTc-RYM1 was radiochemically stable in mouse blood for 5 h and demonstrated rapid renal clearance and lower blood levels in vivo compared with 99mTc-RP805. 99mTc-RYM1 binding to aneurysm and its specificity were shown by autoradiography in carotid aneurysm. Angiotensin II infusion in apoE-/- mice for 4 wk resulted in AAA formation in 36% (4/11) of surviving animals. In vivo 99mTc-RYM1 small-animal SPECT/CT images showed higher uptake of the tracer in AAA than nondilated aortae. Finally, aortic uptake of 99mTc-RYM1 in vivo correlated with aortic MMP activity and CD68 expression. Conclusion: The newly developed pan-MMP inhibitor-based tracer 99mTc-RYM1 displays favorable pharmacokinetics for early vascular imaging and enables specific detection of inflammation and MMP activity in aneurysm.

Matrix Metalloproteinase-Targeted Imaging of Lung Inflammation and Remodeling.

Imaging techniques for detection of molecular and cellular processes that precede or accompany lung diseases are needed. Matrix metalloproteinases (MMPs) play key roles in the development of pulmonary pathology. The objective of this study was to investigate the feasibility of in vivo MMP-targeted molecular imaging for detection of lung inflammation and remodeling. METHODS: Lung-specific IL-13 transgenic (Club cell 10-kDa protein [CC10]-IL-13 Tg) mice and wild-type littermates were used in this study. Lung structure, gene expression, and MMP activity were assessed by histology, real-time reverse transcription polymerase chain reaction, Western blotting, and zymography. MMP activation was imaged by in vivo small-animal SPECT/CT followed by ex vivo planar imaging. Signal specificity was addressed using a control tracer. The correlation between in vivo MMP signal and gene expression was addressed. RESULTS: CC10-IL-13 Tg mice developed considerable pulmonary tissue remodeling and inflammation. CD68, MMP-12, and MMP-13 were significantly higher in CC10-IL-13 Tg lungs. On in vivo small-animal SPECT/CT and ex vivo planar images, the MMP signal was significantly higher in the lungs of CC10-IL-13 Tg mice than wild-type animals. Furthermore, a nonbinding analog tracer showed significantly lower accumulation in CC10-IL-13 Tg lungs relative to the specific tracer. There was a significant correlation between small-animal SPECT/CT-derived MMP signal and CD68 expression in the lungs (r = 0.70, P < 0.01). CONCLUSION: Small-animal SPECT/CT-based MMP-targeted imaging of the lungs is feasible and reflects pulmonary inflammation. If validated in humans, molecular imaging of inflammation and remodeling can potentially help early diagnosis and monitoring of the effects of therapeutic interventions in pulmonary diseases.

ABCB1 2677G>T/A variant enhances chemosensitivity to anti-cancer agents acting on microtubule dynamics through LAMP1 inhibition.

Overexpression of ABCB1 associated with single nucleotide variants in cancers was reported to encode a protein responsible for drug resistance. We studied chemosensitivity-related genes associated with ABCB1 2677G>T/A variant. The associated genes were identified based on the results of the significance analysis of microarray, and then prediction accuracy was evaluated using the prediction analysis of microarray. Functional assay of the selected gene was performed by using siRNA and drug accumulation study. A higher frequency of chemoresistance to microtubule-modulating agents was found in cell lines with wild-type ABCB1 compared to cell lines with 2677G>T/A ABCB1 variant. Based on the pharmacogenetic association study with 2677 variant, we identified seven genes that could predict chemosensitivity to microtubule dynamics modulators. The classification accuracy with these seven genes was 90.0%, and the predicted probability was 0.73. LAMP1 was the only gene that was commonly related to chemosensitivity. LAMP1 expression levels were relatively higher in chemoresistant ABCB1 wild-type compared to chemosensitive polymorphic cells. But, there was no difference in ABCB1 expression levels between the two groups. Following LAMP1 siRNA, chemosensitivity was restored due to increased intracellular drug accumulation in wild type cell line. In conclusion, ABCB1 2677G>T/A variant enhances chemosensitivity on microtubule dynamics through LAMP1 inhibition.

Optical imaging of MMP-12 active form in inflammation and aneurysm.

Matrix metalloproteinase (MMP)-12 plays a key role in the development of aneurysm. Like other members of MMP family, MMP-12 is produced as a proenzyme, mainly by macrophages, and undergoes proteolytic activation to generate an active form. Accordingly, molecular imaging of the MMP-12 active form can inform of the pathogenic process in aneurysm. Here, we developed a novel family of fluorescent probes based on a selective MMP-12 inhibitor, RXP470.1 to target the active form of MMP-12. These probes were stable in complex media and retained the high affinity and selectivity of RXP470.1 for MMP-12. Amongst these, probe 3 containing a zwitterionic fluorophore, ZW800-1, combined a favorable affinity profile toward MMP-12 and faster blood clearance. In vivo binding of probe 3 was observed in murine models of sterile inflammation and carotid aneurysm. Binding specificity was demonstrated using a non-binding homolog. Co-immunostaining localized MMP-12 probe binding to MMP-12 positive areas and F4/80 positive macrophages in aneurysm. In conclusion, the active form of MMP-12 can be detected by optical imaging using RXP470.1-based probes. This is a valuable adjunct for pathophysiology research, drug development, and potentially clinical applications.

Matrix metalloproteinase inhibitor, doxycycline and progression of calcific aortic valve disease in hyperlipidemic mice.

Calcific aortic valve disease (CAVD) is the most common cause of aortic stenosis. Currently, there is no non-invasive medical therapy for CAVD. Matrix metalloproteinases (MMPs) are upregulated in CAVD and play a role in its pathogenesis. Here, we evaluated the effect of doxycycline, a nonselective MMP inhibitor on CAVD progression in the mouse. Apolipoprotein (apo)E(-/-) mice (n = 20) were fed a Western diet (WD) to induce CAVD. After 3 months, half of the animals was treated with doxycycline, while the others continued WD alone. After 6 months, we evaluated the effect of doxycycline on CAVD progression by echocardiography, MMP-targeted micro single photon emission computed tomography (SPECT)/computed tomography (CT), and tissue analysis. Despite therapeutic blood levels, doxycycline had no significant effect on MMP activation, aortic valve leaflet separation or flow velocity. This lack of effect on in vivo images was confirmed on tissue analysis which showed a similar level of aortic valve gelatinase activity, and inflammation between the two groups of animals. In conclusion, doxycycline (100 mg/kg/day) had no effect on CAVD progression in apoE(-/-) mice with early disease. Studies with more potent and specific inhibitors are needed to establish any potential role of MMP inhibition in CAVD development and progression.

Molecular Imaging of Angiogenesis and Vascular Remodeling in Cardiovascular Pathology.

Angiogenesis and vascular remodeling are involved in a wide array of cardiovascular diseases, from myocardial ischemia and peripheral arterial disease, to atherosclerosis and aortic aneurysm. Molecular imaging techniques to detect and quantify key molecular and cellular players in angiogenesis and vascular remodeling (e.g., vascular endothelial growth factor and its receptors, αvß3 integrin, and matrix metalloproteinases) can advance vascular biology research and serve as clinical tools for early diagnosis, risk stratification, and selection of patients who would benefit most from therapeutic interventions. To target these key mediators, a number of molecular imaging techniques have been developed and evaluated in animal models of angiogenesis and vascular remodeling. This review of the state of the art molecular imaging of angiogenesis and vascular (and valvular) remodeling, will focus mostly on nuclear imaging techniques (positron emission tomography and single photon emission tomography) that offer high potential for clinical translation.

The neuropilin-like protein ESDN regulates insulin signaling and sensitivity.

Insulin effects on cell metabolism, growth, and survival are mediated by its binding to, and activation of, insulin receptor. With increasing prevalence of insulin resistance and diabetes there is considerable interest in identifying novel regulators of insulin signal transduction. The transmembrane protein endothelial and smooth muscle cell-derived neuropilin-like protein (ESDN) is a novel regulator of vascular remodeling and angiogenesis. Here, we investigate a potential role of ESDN in insulin signaling, demonstrating that Esdn gene deletion promotes insulin-induced vascular smooth muscle cell proliferation and migration. This is associated with enhanced protein kinase B and mitogen-activated protein kinase activation as well as insulin receptor phosphorylation. Likewise, insulin signaling in the liver, muscle, and adipose tissue is enhanced in Esdn(-/-) mice, and these animals exhibit improved insulin sensitivity and glucose homeostasis in vivo. The effect of ESDN on insulin signaling is traced back to its interaction with insulin receptor, which alters the receptor interaction with regulatory adaptor protein-E3 ubiquitin ligase pairs, adaptor protein with pleckstrin homology and Src homology 2 domain-c-Cbl and growth factor receptor bound protein 10-neuronal precursor cell-expressed developmentally downregulated 4. In conclusion, our findings establish ESDN as an inhibitor of insulin receptor signal transduction through a novel regulatory mechanism. Loss of ESDN potentiates insulin's metabolic and mitotic effects and provides insights into a novel therapeutic avenue.

Multimodality and molecular imaging of matrix metalloproteinase activation in calcific aortic valve disease.

UNLABELLED: Calcific aortic valve disease (CAVD) is the most common cause of aortic stenosis. Matrix metalloproteinases (MMPs) are upregulated in CAVD and contribute to valvular remodeling and calcification. We investigated the feasibility and correlates of MMP-targeted molecular imaging for detection of valvular biology in CAVD. METHODS: Apolipoprotein E-deficient (apoE(-/-)) mice were fed a Western diet (WD) for 3, 6, and 9 mo (n = 108) to induce CAVD. Wild-type mice served as the control group (n = 24). The development of CAVD was tracked with CT, echocardiography, MMP-targeted small-animal SPECT imaging using (99m)Tc-RP805, and histologic analysis. RESULTS: Key features of CAVD­leaflet thickening and valvular calcification­were noted after 6 mo of WD and were more pronounced after 9 mo. These findings were associated with a significant reduction in aortic valve leaflet separation and a significant increase in transaortic valve flow velocity. On in vivo SPECT/CT images, MMP signal in the aortic valve area was significantly higher at 6 mo in WD mice than in control mice and decreased thereafter. The specificity of the signal was demonstrated by blocking, using an excess of nonlabeled precursor. Similar to MMP signal, MMP activity as determined by in situ zymography and valvular inflammation by CD68 staining were maximal at 6 mo. In vivo (99m)Tc-RP805 uptake correlated significantly with MMP activity (R(2) = 0.94, P < 0.05) and CD68 expression (R(2) = 0.98, P < 0.01) in CAVD. CONCLUSION: MMP-targeted imaging detected valvular inflammation and remodeling in a murine model of CAVD. If this ability is confirmed in humans, the technique may provide a tool for tracking the effect of emerging medical therapeutic interventions and for predicting outcome in CAVD.

Imaging vessel wall biology to predict outcome in abdominal aortic aneurysm.

BACKGROUND: Abdominal aortic aneurysm (AAA) rupture risk is currently determined based on size and symptoms. This approach does not address the rupture risk associated with small aneurysms. Given the role of matrix metalloproteinases (MMPs) in AAA weakening and rupture, we investigated the potential of MMP-targeted imaging for detection of aneurysm biology and prediction of outcome in a mouse model of AAA with spontaneous rupture. METHODS AND RESULTS: Fifteen-week-old mice (n=66) were infused with angiotensin II for 4 weeks to induce AAA. Saline-infused mice (n=16) served as control. The surviving animals underwent in vivo MMP-targeted micro-single photon emission computed tomographic/computed tomographic imaging, using RP805, a technetium-99m-labeled MMP-specific tracer, followed by ex vivo planar imaging, morphometry, and gene expression analysis. RP805 uptake in suprarenal aorta on micro-single photon emission computed tomographic images was significantly higher in animals with AAA when compared with angiotensin II-infused animals without AAA or control animals. CD68 expression and MMP activity were increased in AAA, and significant correlations were noted between RP805 uptake and CD68 expression or MMP activity but not aortic diameter. A group of angiotensin II-infused animals (n=24) were imaged at 1 week and were followed up for additional 3 weeks. RP805 uptake in suprarenal aorta at 1 week was significantly higher in mice that later developed rupture or AAA. Furthermore, tracer uptake at 1 week correlated with aortic diameter at 4 weeks. CONCLUSIONS: MMP-targeted imaging reflects vessel wall inflammation and can predict future aortic expansion or rupture in murine AAA. If confirmed in humans, this may provide a new paradigm for AAA risk stratification.

Lipid lowering and imaging protease activation in atherosclerosis.

BACKGROUND: Lipid lowering is a mainstay of modern therapeutic approach to atherosclerosis. We sought to evaluate matrix metalloproteinase (MMP)-targeted microSPECT imaging for tracking of the effect of lipid-lowering interventions on plaque biology in atherosclerotic mice in vivo. METHODS AND RESULTS: ApoE(-/-) mice fed on a high fat diet (HFD) for 2 months were randomly assigned to continuation of HFD, HFD plus simvastatin, HFD plus fenofibrate and high fat withdrawal (HFW). The animals underwent serial microSPECT/CT imaging using RP805, a (99m)Tc-labeled MMP-targeted tracer at 1 and 4 weeks after randomization. All three interventions reduced total blood cholesterol by 4 weeks. In animals on HFD, aortic arch RP805 uptake significantly increased from 1 week to 4 weeks. Tracer uptake in fenofibrate and HFW groups was significantly lower than uptake in the HFD group at 4 weeks. Similarly, CD 68 gene expression, reflecting plaque inflammation, was significantly lower in fenofibrate and HFW groups compared to HFD group. MMP tracer uptake significantly correlated with aortic CD68, but not VE-cadherin or smooth muscle α-actin expression. CONCLUSIONS: MMP tracer uptake paralleled the effect of lipid-lowering interventions on plaque inflammation in atherosclerotic mice. MMP-targeted imaging may be used to track the effect of therapeutic interventions in atherosclerosis.

Syntaxin 16 regulates lumen formation during epithelial morphogenesis.

The formation and maintenance of cell-cell junctions, both under physiological and pathological conditions, requires the targeting and trafficking of junctional proteins. Proteins of the syntaxin (Stx)-family localize to a variety of subcellular membranes and contribute to intracellular transport of cargo by regulating vesicle fusion events at these sites. Unlike plasma membrane localized Stxs, the roles of endosome- and Golgi-localized stx proteins in epithelial morphogenesis are less understood. Here we show that Stx16- an endosome- and Golgi-localized target-membrane soluble N-ethylmaleimide attachment protein receptor (t-SNARE) that plays a role in membrane trafficking between these compartments - is essential for lumen development. In cultured Madin Darby Canine Kidney (MDCK) cells, Stx16 was selectively upregulated as sparsely plated cells attained confluency. Stx16-depleted confluent monolayers consistently showed lower transepithelial resistance than control monolayers, and failed to maintain endogenous and ectopically expressed E-cadherin at the adherens junctions due to decreased recycling. We further found that whereas cysts formed by MDCK cells cultured in Matrigel have a single hollow lumen, those formed by stx16-depleted counterparts had multiple lumens, due to abnormal orientiation of the mitotic spindle. Finally, a similar role for stx16 function in vivo is indicated by our analysis of pronephric-duct development in zebrafish expressing the claudinB:lynGFP transgene; lack of stx16 function in this structure (in stx16-morphant embryos) led to the development of enlarged, torturous pronephric ducts with more than one lumen. Taken together, our in vitro and in vivo studies establish a role for Stx16 in maintaining the integrity of cell-cell junctions, and thereby in morphogenesis of the kidney epithelial lumen.

The myosin motor Myo1c is required for VEGFR2 delivery to the cell surface and for angiogenic signaling.

Vascular endothelial growth factor receptor-2 (VEGFR2) is a receptor tyrosine kinase that is expressed in endothelial cells and regulates angiogenic signal transduction under both physiological and pathological conditions. VEGFR2 turnover at the plasma membrane (PM) is regulated by its transport through endocytic and secretory transport pathways. Short-range cargo trafficking along actin filaments is commonly regulated by motor proteins of myosin superfamily. In the current study, performed in primary human endothelial cells, we demonstrate that unconventional myosin 1c (Myo1c; class I family member) regulates the localization of VEGFR2 at the PM. We further demonstrate that the recruitment of VEGFR2 to the PM and its colocalization with Myo1c and caveolin-1 occur in response to VEGF-A (VEGF) stimulation. In addition, VEGF-induced delivery of VEGFR2 to the cell surface requires Myo1c; surface VEGFR2 levels are reduced in the absence of Myo1c and, more importantly, are restored by the overexpression of wild-type but not mutant Myo1c. Subcellular density gradient fractionation revealed that partitioning of VEGFR2 into caveolin-1- and Myo1c-enriched membrane fractions is dependent on VEGF stimulation. Myo1c depletion resulted in increased VEGF-induced VEGFR2 transport to the lysosomes for degradation and was rescued by applying either brefeldin A, which blocks trafficking between the endoplasmic reticulum and the Golgi complex, or dynasore, an inhibitor of dynamin-mediated endocytosis. Myo1c depletion also reduced VEGF-induced VEGFR2 phosphorylation at Y1175 and phosphorylation-dependent activation of ERK1/2 and c-Src kinase, leading to reduced cell proliferation and cell migration. This is the first report demonstrating that Myo1c is an important mediator of VEGF-induced VEGFR2 delivery to the cell surface and plays a role in angiogenic signaling.