Theranostic immunoliposomes for osteoarthritis.

Although there have been substantial advancements in the treatment of inflammatory arthritis, treatments for osteoarthritis (OA) have lagged and currently are primarily palliative until joints become totally dysfunctional and prosthetic replacement is needed. One obstacle for developing a preventive therapy for OA is the lack of good tools for efficiently diagnosing the disease and monitoring its progression during the early stages when the effect of therapeutic drugs or biologics is most likely to be effective. We have developed near infrared immunoliposomes conjugated with type II collagen antibody for diagnosis and treatment of early OA. These immunoliposomes bind to damaged but not normal cartilage. Utilizing these reagents, we can quantitate exposure of type II collagen during cartilage degradation in individual joints in vivo in a guinea pig. Immunoliposomes could be used to determine the effectiveness of therapeutic interventions in small animals as well as vehicles for localized drug delivery to OA chondrocytes. FROM THE CLINICAL EDITOR: This team of authors have developed near infrared immunoliposomes conjugated with type II collagen antibody for diagnosis and treatment of early OA, with promising results demonstrated in a guinea pig model.

Endothelial protein kinase C isoform identity and differential activity of PKCzeta in an athero-susceptible region of porcine aorta.

Endothelial protein kinase C (PKC) signaling was investigated in different regions of normal porcine aorta. The locations map to differential atherosclerotic susceptibility and correlate with sites of disturbed (DF) or undisturbed (UF) local flow profiles. Endothelial lysates were isolated from the inner curvature of the aortic arch (DF; athero-susceptible) and a nearby UF region of the descending thoracic aorta (UF; athero-protected), and in some experiments a distant athero-protected UF site, the common carotid artery. Total endothelial PKC activity in the DF regions was 145% to 240% of that in both UF locations (P<0.05), whereas the UF regions were not significantly different from each other. PKC protein isoforms alpha, beta, epsilon, iota, lambda, and zeta were expressed in similar proportions in both aortic regions, suggesting that differences of kinase activity were not directly attributable to expression levels. Inhibition of members of the "conventional" and "novel" PKC families had no differential effect on regional kinase activity. However, inhibition of PKCzeta, a member of the "atypical" PKC family, reduced the DF lysate kinase activity to that of UF levels (NS P=0.35). Differential phosphorylation of PKCzeta Thr410 and Thr560, along with increased levels of PKCzeta degradation products in UF endothelial lysates, suggested posttranslational modification of PKCzeta as the basis for site-specific differences in vivo. Steady-state regional heterogeneity of an important family of regulatory proteins in intact arterial endothelium in vivo may link localized athero-susceptibility and the associated hemodynamic environment.

Matrix metalloproteinase 9 facilitates collagen remodeling and angiogenesis for vascular constructs.

Degradation of the extracellular matrix, facilitated by matrix metalloproteinases (MMPs), can lead to mechanical failure of vascular constructs, suggesting that MMP inhibition could improve survival of constructs. Therefore, we investigated the role of MMP-9 in collagen remodeling in vitro, focusing on the three major steps of production, degradation, and organization. Because an adequate blood supply is essential for survival of tissue-engineered constructs, we also evaluated the influence of MMP-9 deficiency on angiogenesis in vivo by implantation of thin biodegradable polymer scaffolds. Using aortic smooth muscle cells (SMCs) from wild-type and genetically deficient (9KO) mice, we examined the role of MMP-9 in collagen mRNA expression and protein accumulation, both with and without ascorbic acid treatment. We measured collagen assembly in a fibrillogenesis assay. We investigated in vivo angiogenesis and cell invasion, using fluorescence microangiography and histology. MMP-9 deficiency did not affect collagen mRNA production or polymer scaffold degradation, but collagen accumulation was greater in cultures of 9KO SMCs than in wild-type SMCs. Both MMP-9 deficiency and chemical inhibition impaired collagen degradation. Ascorbic acid treatment enhanced collagen production by 9KO SMCs compared with wild-type SMCs at 3 days, but by 7 days this effect was reversed. MMP-9 improved fibrillogenesis of collagen, significantly more on ascorbic acid treatment. MMP-9 deficiency dramatically decreased inflammatory cell invasion, but also capillary formation within biodegradable polymer scaffolds in vivo. Our data suggest that MMP inhibition, by impairing collagen organization and angiogenesis, might have detrimental effects on the survival of vascular constructs.

Vascular oxidant stress enhances progression and angiogenesis of experimental atheroma.

BACKGROUND: Although multiple pathological processes have been associated with oxidative stress, the causative relation between oxidative stress and arterial lesion progression remains unclear. METHODS AND RESULTS: To test the effect of creating arterial wall oxidative stress, we compared progression of mouse carotid lesions induced by flow cessation in the wild-type (WT) versus transgenic mice (Tg(p22vsmc)), in which overexpression of p22phox, a critical component of NAD(P)H oxidase was targeted to smooth muscle cell (SMC). Compared with WT mice, arterial lesions grew significantly larger in Tg(p22vsmc) (P<0.001) and demonstrated elevated hydrogen peroxide (H2O2) and vascular endothelial growth factor (VEGF) levels at all time points examined (P<0.001, n=4 animals per time point), probably related to increased expression of hypoxia inducible factor (HIF)-1alpha via SMC oxidative stress in the Tg(p22vsmc) arteries, both basally (203+/-12% versus WT, P<0.001, n=3) and after lesion formation. Interestingly, Tg(p22vsmc) lesions were complicated by extensive neointimal angiogenesis. In vitro experiments confirmed SMCs isolated from Tg(p22vsmc) to be the source for increased H2O2, VEGF, and HIF-1alpha and their capacity to induce angiogenic cord-like structures when cocultured with endothelial cells. The antioxidant ebselen inhibited SMC activities in vitro and intralesion angiogenesis and lesion progression in vivo. CONCLUSIONS: We have demonstrated a novel pathway by which oxidative stress can trigger in vivo an angiogenic switch associated with experimental plaque progression and angiogenesis. This pathway may be related to human atheroma progression and destabilization through intraplaque hemorrhage.

Optimization of isolation and functional characterization of primary murine aortic endothelial cells.

In this study the authors sought to improve the technique of isolating and culturing murine aortic endothelial cells (MAECs) for future use in studying endothelium-specific roles in complex pathologies such as atherosclerosis. The authors utilized a transgenic mouse expressing green fluorescent protein (GFP) under control of the endothelial specific Tie-2 promoter, allowing the tracking of endothelial cells throughout the isolation and purification. Briefly, aortas were harvested, the adventitia was removed, and strips were placed lumen side down into Matrigel. After 14 days, endothelial cells were isolated from the total cell population by fluorescence-activated cell sorting (FACS) using the GFP signal. The authors confirmed the endothelial phenotype of sorted cells based upon endothelial-specific Griffonia simplicifolia lectin staining, uptake of acetylated low-density lipoprotein (LDL), and von Willebrand factor (vWF) and VE-cadherin staining. The authors also confirmed the cells' ability to form tubes in Matrigel, and for cellular alignment with flow to occur following monolayer culture under unidirectional laminar shear stress but not following culture under oscillatory flow. Although GFP fluorescence--based vital sorting was used initially to optimize the isolation, the authors have verified that this method is applicable to the isolation of MAECs from other strains and backgrounds of mice through the use of other endothelial markers.

Expression of matrix metalloproteinase-9 in endothelial cells is differentially regulated by shear stress. Role of c-Myc.

Atherosclerotic plaques preferentially localize to areas of the vasculature with complex laminar or oscillatory blood flow. Prior data implicate matrix metalloproteinases (MMPs) in the initiation and progression of atherosclerotic lesions. In cultured endothelial cells, oscillatory but not unidirectional shear significantly increases MMP-9 mRNA as well as secretion of the MMP-9 protein (p < 0.05). In contrast, cell-associated protein levels of Tissue Inhibitor of MMP 1 (TIMP-1), an inhibitor of MMP-9, are insensitive to the shear regimen. To investigate transcriptional regulation of MMP-9 gene expression, we utilized retroviral-based reporter constructs containing different lengths of the human MMP-9 promoter. The activity of the full MMP-9 promoter is 3-fold higher (p < 0.05) in unidirectional shear compared with static conditions, and the activity is further increased approximately 10-fold by oscillatory shear (p < 0.01) over unidirectional flow. Our data identify a shear-sensitive binding site at -152 in the MMP-9 promoter. We show that the c-Myc transcription factor binds specifically to this site and that reporter constructs in which the c-Myc binding site was abolished lacked the shear responsiveness of native MMP-9 reporter constructs. Our results suggest that endothelial MMP-9 expression is flow-sensitive and is up-regulated by oscillatory flow via activation of c-Myc. This effect may contribute to the development and progression of atherosclerotic lesions in areas of vasculature that are subject to disturbed flow.

Uniaxial strain upregulates matrix-degrading enzymes produced by human vascular smooth muscle cells.

Arteries remodel in response to environmental changes. We investigated whether mechanical strain modulates production of matrix metalloproteinase (MMP)-2 and -9 by cultured vascular smooth muscle cells (SMC). MMP-2 and MMP-9 expression were tested using human saphenous vein SMC cultured on silicone membranes at rest or subjected to physiological levels (5%) of stationary or cyclical (1 Hz) uniaxial strain. Compared with control, stationary strain significantly increased MMP-2 mRNA levels at all time points, whereas cyclic strain decreased it after 48 h. Both secreted and cell-associated pro-MMP-2 levels were increased by stationary strain at all times (P < 0.01), whereas cyclic strain decreased secreted levels after 48 h (P < 0.02). MMP-9 mRNA levels and pro-MMP-9 protein were increased after 48 h of stationary stretch (P < 0.01) compared with both no strain and cyclic strain. Our study indicates that vascular SMC show a selective response to different types of strain. We suggest that local increases in stationary mechanical strain resulting from stenting, hypertension, or atherosclerosis may lead to enhanced matrix degradation by SMC.

Targeted disruption of the matrix metalloproteinase-9 gene impairs smooth muscle cell migration and geometrical arterial remodeling.

Matrix remodeling plays an important role in the physiological and pathological remodeling of blood vessels. We specifically investigated the role of matrix metalloproteinase (MMP)-9, an MMP induced during arterial remodeling, by assessing the effects of genetic MMP-9 deficiency on major parameters of arterial remodeling using the mouse carotid artery flow cessation model. Compared with remodeling of matched wild-type (WT) arteries, MMP-9 deficiency decreased intimal hyperplasia, reduced the late lumen loss, eliminated the correlation between intimal hyperplasia and geometric remodeling, and led to significant accumulation of interstitial collagen. Biochemical analysis of MMP-9 knockout (KO) arterial tissue and isolated smooth muscle cells (SMCs) confirmed the lack of MMP-9 expression or compensation by other gelatinases. To investigate potential mechanisms for the in vivo observations, we analyzed in vitro effects of MMP-9 deficiency on the migration, proliferation, and collagen gel contracting capacity of aortic SMCs isolated from MMP-9 KO and WT mice. Although proliferation was comparable, we found that MMP-9-deficient cells had not only decreased migratory activity, but they also had decreased capacity to contract collagen compared with WT cells. Thus, MMP-9 appears to be involved not only in degradation, but also in reorganization of a collagenous matrix, both facets being essential for the outcome of arterial remodeling. Our results also establish MMP-9 as an attractive therapeutic target for limiting the effects of pathological arterial remodeling in restenosis and atherosclerosis.

Expansive arterial remodeling is associated with increased neointimal macrophage foam cell content: the murine model of macrophage-rich carotid artery lesions.

BACKGROUND: Recent observations associate plaque instability with expansive arterial remodeling, suggesting a common driving mechanism. METHODS AND RESULTS: To demonstrate that macrophages, a characteristic of vulnerable plaques, also assist in expansive remodeling, we compared carotid artery remodeling due to formation of experimental macrophage-rich and macrophage-poor lesions in the flow cessation model in hypercholesterolemic apolipoprotein E knockout (ApoE KO) and wild type (WT) mice. After ligation, macrophages started to rapidly accumulate in ApoE KO but not in WT carotid artery lesions. Macrophage-rich ApoE KO intimal lesions grew fast, typically occluding within 14 days, despite a tripling of the vessel area. Outward remodeling of macrophage-rich ApoE KO arteries positively correlated with macrophage area (r2=0.600, P<0.001). To investigate potential mechanisms of macrophage-enabled expansive remodeling, we compared levels of matrix metalloproteinases in homogenates of macrophage-rich and macrophage-poor carotid arteries. Gelatinolytic activity of macrophage-rich lesions increased faster and reached maximal levels several fold higher than in the macrophage-poor WT lesions. CONCLUSIONS: Our results suggest that macrophages facilitate expansive arterial remodeling through increased matrix degradation by matrix metalloproteinases. This initially favorable remodeling action may eventually increase the vulnerability of macrophage-rich atherosclerotic plaques.