Performance of a new fluorescence‐labeled MMP inhibitor to image tumor MMP activity in vivo in comparison to an MMP‐activatable probe

Matrix metalloproteinases (MMPs) are proteolytic enzymes that degrade structural components of the extracellular matrix and participate in pathologies such as cancer and inflammatory disorders. The development of novel contrast agents for optical imaging of MMP activity in vivo is of great interest. The commonly used near‐infrared fluorescence‐compatible agents are dye‐quenched probes that do not emit fluorescence until they interact with MMPs. In contrast, fluorescent synthetic low‐molecular‐weight MMP inhibitors have not been systematically employed. The aim of this study was to evaluate the performance of our recently developed Cy5.5‐labeled MMP inhibitor to image MMP activity in tumors in vivo compared with activatable fluorescent MMP‐sensing probes. The dynamic uptake of Cy5.5‐AF489 into four different tumor entities was analyzed in xenografted mice by intravenous injection and subsequent fluorescence reflectance imaging. Tumors were characterized in regard to their MMP‐2 and ?9 mRNA expressions (qRT‐PCR analysis), proteins (immunohistochemistry) and gelatinase/collagenase activities (in situ zymography). Cy5.5‐AF489 was compared with MMPSense^TM 680 and MMPSense^TM 750 FAST, two commercially available MMP‐activatable probes. Cy5.5‐AF489 showed a specific tumor uptake, which was blocked by pre‐injection of the unlabeled MMPI, and discriminated between tumors with high or low MMP‐2/‐9 expressions. Our optical probe facilitated faster visualization of MMP‐active tumors accompanied by excellent tumor‐to‐background ratios when compared with activatable probes. The MMP inhibitor Cy5.5‐AF489 permits fast in vivo imaging of MMP expression/activity in tumors. Given its small molecular weight and non‐peptidic structure, translational imaging from a preclinical application to a diagnostic tool for MMP‐related diseases seems feasible. Copyright © 2012 John Wiley & Sons, Ltd.     

In vivo biodistribution of radiolabeled MMP‐2/9 activatable cell‐penetrating peptide probes in tumor‐bearing mice

Matrix metalloproteinases (MMPs) play a pivotal role in cancer progression and present therefore an interesting biomarker for early diagnosis, staging and therapy evaluation. Consequently, MMP‐specific molecular imaging probes have been proposed for noninvasive visualization and quantification of MMP activity. An interesting approach is MMP‐2/9 activatable cell‐penetrating peptides (ACPP) that accumulate in the tumor tissue after activation. However, a recent study revealed that probe activation occurred already in the vasculature followed by nonspecific tumor targeting. In the latter study, biodistribution was determined 6 and 24 h post‐ACPP injection. An alternative explanation could still be that the kinetics of tumor‐specific activation is faster than that of blood activation plus subsequent nonspecific uptake in tumor. The aim of this study was to assess if tumor‐specific ACPP activation occurs in mice with MMP‐2/9 positive subcutaneous HT‐1080 tumors at 3 h post‐injection. As control, we studied the MMP‐2/9 sensitive ACPP in mice bearing subcutaneous BT‐20 tumors with low MMP‐2/9 expression to test if probe cleavage correlates with tumoral MMP expression. Ex vivo biodistribution showed no improved tumoral ACPP activation in HT‐1080 tumor‐bearing mice at 3 h post‐injection compared with previous reported data collected at 24 h post‐injection. Furthermore, tumoral uptake and relative tumoral activation for ACPP were similar in both BT‐20 and HT‐1080 tumor‐bearing mice. In conclusion, this study suggests that tumoral ACPP uptake in these tumor models originates from probe activation in the vasculature instead of tumor‐specific MMP activation. Novel ACPPs that target tissue‐specific proteases without nonspecific activation may unleash the full potential of the elegant ACPP concept. Copyright © 2014 John Wiley & Sons, Ltd.     

Site‐specific tissue inhibitor of metalloproteinase‐1 governs the matrix metalloproteinases‐dependent degradation of crosslinked collagen scaffolds and is correlated with interleukin‐10

We have previously shown that the foreign body reaction (FBR) against crosslinked collagen type I (Col‐I) differs between subcutaneous and epicardial implantation sites; Col‐I was quickly degraded epicardially, whereas degradation was attenuated subcutaneously. The current study set out to dissect the nature and regulation of the MMP‐based degradation of implanted Col‐I in mice during the FBR. Immunohistochemistry showed that MMP‐2, MMP‐8 and MMP‐13 were present in subcutaneous and epicardial implants, whereas only MMP‐9 was also present epicardially. Western blotting showed that MMP‐8 and MMP‐9 were mainly present in their inactive proform. In contrast, collagenase MMP‐13 and gelatinase MMP‐2 were the predominant active MMPs at both sites. Interestingly, the major MMP inhibitor TIMP‐1 was solely observed in subcutaneous implants, which is why MMP‐13 and MMP‐2 are not able to degrade the collagen scaffold at the subcutaneous implantation site. Interleukin 10 (IL‐10), a potent inducer of TIMP‐1 expression, was also mainly detected subcutaneously; giant cells were the main source. Therefore, we surmise that IL‐10, through regulation of the balance between MMPs and TIMP‐1, suppresses the FBR against implanted biomaterials. Together, our findings would provide cues and clues to improve future therapies in regenerative medicine that are based on the tuned regulation of the degradation of biomaterial scaffolds. Copyright © 2010 John Wiley & Sons, Ltd.     

A comprehensive review on controls in molecular imaging: lessons from MMP‐2 imaging

Metalloproteinases (MMPs), including MMP‐2, play critical roles in tissue remodeling and are involved in a large array of pathologies, including cancer, arthritis and atherosclerosis. Their prognostic value warranted a large investment or resources in the development of noninvasive detection methods, based on probes for many current clinical and pre‐clinical imaging modalities. However, the potential of imaging techniques is only matched by the complexity of the data they generate. This complexity must be properly assessed and accounted for in the early steps of probe design and testing in order to accurately determine the efficacy and efficiency of an imaging strategy. This review proposes basic rules for the evaluation of novel probes by addressing the specific case of MMP targeted probes. Copyright © 2014 John Wiley & Sons, Ltd.     

Novel Gd(III)‐based probes for MR molecular imaging of matrix metalloproteinases

Two novel Gd‐based contrast agents (CAs) for the molecular imaging of matrix metalloproteinases (MMPs) were synthetized and characterized in vitro and in vivo. These probes were based on the PLG*LWAR peptide sequence, known to be hydrolyzed between Gly and Leu by a broad panel of MMPs. A Gd–DOTA chelate was conjugated to the N‐terminal position through an amide bond, either directly to proline (compd Gd–K11) or through a hydrophilic spacer (compd Gd–K11N). Both CA were made strongly amphiphilic by conjugating an alkyl chain at the C‐terminus of the peptide sequence. Gd–K11 and Gd–K11N have a good affinity for β‐cyclodextrins (K_D 310 and 670 µ m respectively) and for serum albumin (K_D 350 and 90 µ m respectively), and can be efficiently cleaved in vitro at the expected site by MMP‐2 and MMP‐12. Upon MMP‐dependent cleavage, the CAs lose the C‐terminal tetrapeptide and the alkyl chain, thus undergoing to an amphiphilic‐to‐hydrophilic transformation that is expected to alter tissue pharmacokinetics. To prove this, Gd–K11 was systemically administered to mice bearing a subcutaneous B16.F10 melanoma, either pre‐treated or not with the broad spectrum MMP inhibitor GM6001 (Ilomastat). The washout of the Gd‐contrast enhancement in MR images was significantly faster for untreated subjects (displaying MMP activity) with respect to treated ones (MMP activity inhibited). The washout kinetics of Gd‐contrast enhancement from the tumor microenvironment could be then interpreted in terms of the local activity of MMPs. Copyright © 2012 John Wiley & Sons, Ltd.     

CD40 ligand shedding is regulated by interaction between matrix metalloproteinase‐2 and platelet integrin αIIbβ3

Summary. Background: CD40 ligand (CD40L, CD154) in the circulatory system is mainly contained in platelets, and surface‐expressed CD40L on activated platelets is subsequently cleaved by proteolytic activity to generate soluble CD40L (sCD40L). However, the enzyme responsible for the shedding of CD40L in activated platelets has not been clearly identified yet. We have recently found that molecular interaction of matrix metalloproteinase‐2 (MMP‐2) with integrin α_IIbβ₃ is required for the enhancement of platelet activation. Objectives: To elucidate the biochemical mechanism of MMP‐2‐associated sCD40L release. Methods: Localization of MMP‐2 and CD40L in platelets was analyzed by flow cytometry and fluorescence microscopy. The release of sCD40L from activated platelets was measured by enzyme‐linked immunosorbent assay. MMP‐2 binding to α_IIbβ₃ was analyzed by immunoprecipitation and western blotting. Recombinant hemopexin‐like domain and MMP‐2‐specific inhibitor were used to characterize the nature of MMP‐2 binding and catalytic activity. Results: It was revealed that interaction of MMP‐2 with α_IIbβ₃ is required for effective production of sCD40L in activated human platelets. Platelet activation and release of sCD40L were significantly affected by inhibition of platelet‐derived MMP‐2 activity or by inhibition of binding between the enzyme and the integrin. It was also found in platelet‐rich plasma that MMP‐2 activity is responsible for generating sCD40L. Conclusions: The results presented here strongly suggest that MMP‐2 interacts with α_IIbβ₃ to regulate the shedding of CD40L exposed on the surfaces of activated human platelets.     

Evaluation of metalloproteinases 2 and 9 and their inhibitors in physiologic and pre‐eclamptic pregnancy

Matrix metalloproteinases (MMPs) are a family of zinc and calcium‐dependent endopeptidases involved in remodeling and physiological homeostasis of extracellular matrix (ECM). The metalloproteinases activity is predominantly modulated by specific tissue inhibitors of matrix metalloproteinases (TIMPs). The balance between MMPs and TIMPs is likely to play an important role in remodeling uterine arteries in pregnancy, and it may represent means by which vasodilatation is maintained in later pregnancy. Moreover, increased levels of MMPs and in particular MMP‐2 play a role in the vascular alterations induced by hypertension. The aim of this study was the evaluation of MMP‐2 and ‐9, along with their inhibitors TIMP‐1 and ‐2, in pre‐eclamptic women compared with normotensive pregnancy and non‐pregnant women. Fourteen pre‐eclamptic women were compared with 37 normotensive women in different gestational age and 21 non‐pregnant women. Multiplexed sandwich enzyme‐linked immunosorbent assay was used to measure MMPs and TIMPs simultaneously. MMP‐2 levels were significantly higher in pre‐eclamptic women vs. both non‐pregnant and physiologic pregnant women. MMP‐9 concentrations were significantly higher in physiologic pregnant vs. non‐pregnant women. The serum levels of TIMP‐1 were significantly higher in pre‐eclamptic vs. both non‐pregnant and physiologic pregnant women. TIMP‐2 values were higher in physiologic pregnant women and pre‐eclamptic women vs. non‐pregnant women. A positive correlation between MMP‐9 values and gestational age was observed in normal pregnant women. Results of the present investigation confirm that MMP‐2 and TIMP‐1 values are significantly higher in preeclampsia. We confirm that the modification of the fine balance between MMPs and their inhibitors plays a greater role in the structural and functional vascular changes of women with complicated pregnancies. J. Clin. Lab. Anal. 23:88–92, 2009. © 2009 Wiley‐Liss, Inc.     

Immunohistochemical determination of the extracellular matrix modulation in a rat model of choline‐deprived myocardium: the effects of carnitine

Choline has been identified as an essential nutrient with crucial role in many vital biological functions. Recent studies have demonstrated that heart dysfunction can develop in the setting of choline deprivation even in the absence of underlying heart disease. Matrix metalloproteinases (MMPs) are responsible for extracellular matrix degradation, and the dysregulation of MMP‐2 and MMP‐9 has been involved in the pathogenesis of various cardiovascular disorders. The aim of the study was to investigate the role of MMPs and their inhibitors (TIMPs), in the pathogenesis of choline deficiency‐induced cardiomyopathy, and the way they are affected by carnitine supplementation. Male Wistar Albino adult rats were divided into four groups and received standard or choline‐deficient diet with or without L‐carnitine in drinking water (0.15% w/v) for 1 month. Heart tissue immunohistochemistry for MMP‐2, MMP‐9, TIMP‐1, and TIMP‐2 was performed. Choline deficiency was associated with suppressed immunohistochemical expression of MMP‐2 and an increased expression of TIMP‐2 compared to control, while it had no impact on TIMP‐1. MMP‐9 expression was decreased without, however, reaching statistical significance. Carnitine did not affect MMP‐2, MMP‐9, TIMP‐1 or TIMP‐2 expression. The pattern of TIMP and MMP modulation observed in a choline deficiency setting appears to promote fibrosis. Carnitine, although shown to suppress fibrosis, does not seem to affect MMP‐2, MMP‐9, TIMP‐1 or TIMP‐2 expression. Further studies will be required to identify the mechanism underlying the beneficial effects of carnitine.     

Pro‐elastogenic effects of bone marrow mesenchymal stem cell‐derived smooth muscle cells on cultured aneurysmal smooth muscle cells

Abdominal aortic aneurysms (AAAs) involve slow proteolysis and loss of structural matrix components (collagen and elastin), which lead to wall thinning, weakening and ultimate rupture. At this time, no established non‐surgical therapy is available to slow or arrest AAA growth. Inhibiting matrix metalloproteases (MMPs; e.g. MMP2 and ?9) overexpressed within AAAs is insufficient to arrest AAA growth, since resident smooth muscle cells (SMCs) are poorly elastogenic and cannot overcome elastolysis to reinstate a healthy elastic matrix. Towards overcoming this limitation, this first study sought to determine the utility of rat bone marrow mesenchymal stem cell (BM‐MSC)‐derived SMCs to stimulate elastin and elastic matrix synthesis and assembly by aneurysmal SMCs (EaRASMCs). BM‐MSCs were successfully differentiated into cells of an SMC lineage (SMLCs). Our study indicates that BM‐MSC‐derived SMLCs secrete trophic factors, contained in conditioned medium (CM) from their cultures, that, when exposed to EaRASMC cultures in real time, stimulate elastin precursor and matrix deposition and crosslinking by these elastogenically deficient cells, with added benefits in terms of attenuating MMPs, specifically MMP9. The results thus lend support to a proposed cell therapy for AAAs, based on the use of BM‐MSC‐derived SMLCs. Although we observed no particular improvement in elastic fibre formation, no attenuation of MMP2 activity and increase in amounts of active MMP2 enzyme, we believe that this study justifies follow‐up studies to improve upon these outcomes. Future studies will explore the effects of concentrated CM collected from long‐term SMLC cultures on EaRASMCs and also investigate the elastogenic output of SMLCs themselves. Copyright © 2014 John Wiley & Sons, Ltd.     

O‐Phenanthroline as modulator of the hypoxic and catabolic response in cartilage tissue‐engineering models

Hypoxia has been shown to be important for maintaining cartilage homeostasis as well as for inducing chondrogenic differentiation. Ensuring low oxygen levels during in vitro culture is difficult, therefore we assessed the chondro‐inductive capabilities of the hypoxia‐mimicking agent O‐phenanthroline, which is also known as a non‐specific matrix metalloproteinase (MMP) inhibitor. We found that O‐phenanthroline reduced the expression of MMP3 and MMP13 mRNA levels during chondrogenic differentiation of human chondrocytes (hChs), as well as after TNFα/IL‐1β exposure in an explant model. Interestingly, O‐phenanthroline significantly inhibited matrix degradation in a TNFα/IL‐1β‐dependent model of cartilage degeneration when compared to control and natural hypoxia (2.5% O₂). O‐Phenanthroline had limited ability to improve the chondrogenic differentiation or matrix deposition in the chondrogenic pellet model. Additionally, O‐phenanthroline alleviated MMP‐induced cartilage degradation without affecting chondrogenesis in the explant culture. The data presented in this study indicate that the inhibitory effect of O‐phenanthroline on MMP expression is dominant over the hypoxia‐mimicking effect. Copyright © 2014 John Wiley & Sons, Ltd.     

Associations of IgG N‐linked oligosaccharide chains and proteases in sera of prostate cancer patients with and without α2‐macroglobulin deficiency

We previously reported on a number of cases of metastatic prostate cancer (PCa) in which serum α2‐macroglobulin (α2M) levels were markedly decreased to less than 20 mg/dl (α2M deficiency). All PCa patients with α2M deficiency had multiple bone metastases. Proteases in ten PCa patients with and without α2M deficiency were studied and compared against ten healthy controls in order to elucidate the relationships between changes in sugar chain structure and neoplasia. We assessed the relationship between ratios of Fr4 to Fr1 and Fr2 (Fr4/Fr1+Fr2 ratios) of oligosaccharide chains, and ratios of free prostate‐specific antigen (PSA) to total PSA (F/T ratios), and serum levels of matrix‐metalloproteinase‐2 (MMP‐2) in PCa progression. Measurement of serum α2M concentration was performed by laser nephelometry. Serum PSA and MMP‐2 levels were determined by enzyme immunoassay and free PSA by radioimmunoassay. N‐linked oligosaccharides of human serum immunoglobulin G were analyzed using fluorophore‐associated carbohydrate electrophoresis. In those PCa patients with α2M deficiency: (a) serum α2M and F/T ratios were lower (P<0.05) and (b) Fr4/Fr1+Fr2 ratios and serum MMP‐2 levels were higher when compared with those PCa patients without α2M deficiency. There was a significant correlation between Fr4/Fr1+Fr2 ratios and F/T ratios or serum MMP‐2 levels in PCa with α2M deficiency (P<0.05). Therefore, these markers may serve as an auxiliary serum tumor marker for monitoring of the bone metastases or progression of disease in PCa. J. Clin. Lab. Anal. 23:125–131, 2009. © 2009 Wiley‐Liss, Inc.     

Polymorphism in dural arteriovenous fistula: matrix metalloproteinase‐2‐1306 C/T as a potential risk factor for sinus thrombosis

Essentials

• Sinus thrombosis may play a crucial role in development of dural arteriovenous fistula (DAVF).
• Little is known about the association between gene polymorphism and the development of DAVF.
• MMP‐2‐1306 C/T showed a higher prevalence rate in DAVF cases with sinus thrombosis.
• MMP‐2‐1306C/T polymorphism is likely a potential risk factor for sinus thrombosis in DAVF.

Summary

Background

Dural arteriovenous fistula (DAVF) is a rare but important cerebrovascular disorder in adults. Little is known about the molecular genetic pathogenesis underlying DAVF development.

Objectives

To investigate the associations of gene polymorphisms and DAVF.

Materials and Methods

By the use of real‐time PCR genotyping, seven single‐nucleotide polymorphisms (SNPs) of angiogenesis‐related genes were analyzed in 72 DAVF patients. Pertinent clinical and imaging data were subgrouped on the basis of location (cavernous sinus versus lateral sinus), lesions (single versus multiple), cerebral venous reflux (CVR) grading (Borden I versus Borden II/III), and sinus thrombosis (with versus without).

Results

We found that individuals carrying the polymorphic allele of matrix metalloproteinase (MMP)‐2‐1306 C/T (rs243865) had a significantly increased risk of sinus thrombosis in DAVF (odds ratio 6.2; 95% confidence interval 1.7–22.9). There was a weak difference in associations of tissue inhibitor of metalloproteinase (TIMP)‐2 (rs2277698) gene polymorphism and DAVF patients subgrouped by CVR grading.

Conclusions

These preliminary results indicate that MMP‐2‐1306 C/T, but not MMP‐9, TIMP‐1, TIMP‐2, and vascular endothelial growth factor A SNP variants, is a risk factor for the development of sinus thrombosis in DAVF patients.

     

Temporal effects of cytokine treatment on lubricant synthesis and matrix metalloproteinase activity of fibroblast‐like synoviocytes

Fibroblast‐like synoviocytes (FLS) are major contributors to the composition and function of synovial fluid (SF). In disease, changes to important SF molecules such as hyaluronic acid (HA), lubricin, and numerous inflammatory markers contribute to a loss of SF functional properties. Previous studies characterized the ability of FLS to produce SF molecules in short‐term cultures using continuous cytokine supplementation. This study assessed the HA, lubricin, and matrix metalloproteinase‐2 (MMP‐2) secretion profile of FLS over 12 days of culture. FLS were subjected to continuous, intermittent, and sequential cytokine treatments of interleukin‐1 beta (IL‐1β), tumour necrosis factor‐alpha (TNF‐α), and transforming growth factor‐beta 1 (TGF‐β1). HA was assessed by an enzyme‐linked immunosorbent assay (ELISA) for content and agarose gel electrophoresis for molecular weight distribution. Relative lubricin content was determined by western blot. Pro MMP‐2 and active MMP‐2 were quantified by gelatin zymography. All intermittent and sequential treatments significantly increased secretion of high‐molecular‐weight (>3 MDa) HA for the duration of the culture. Sequentially treated groups elevated lubricin synthesis, whereas only groups receiving IL‐1β and TNF‐α for 2 days followed by TGF‐β1 for 1 day reduced active MMP‐2 to unstimulated control levels. These data provide important information on the long‐term functional potential of cytokine‐stimulated FLS and suggest that temporal regulation of cytokine exposure can be a powerful tool to guide healthy synovial secretions.     

Three‐dimensional culture of single embryonic stem‐derived neural/stem progenitor cells in fibrin hydrogels: neuronal network formation and matrix remodelling

In an attempt to improve the efficacy of neural stem/progenitor cell (NSPC) based therapies, fibrin hydrogels are being explored to provide a favourable microenvironment for cell survival and differentiation following transplantation. In the present work, the ability of fibrin to support the survival, proliferation, and neuronal differentiation of NSPCs derived from embryonic stem (ES) cells under monolayer culture was explored. Single mouse ES‐NSPCs were cultured within fibrin (fibrinogen concentration: 6 mg/ml) under neuronal differentiation conditions up to 14 days. The ES‐NSPCs retained high cell viability and proliferated within small‐sized spheroids. Neuronal differentiation was confirmed by an increase in the levels of βIII‐tubulin and NF200 over time. At day 14, cell‐matrix constructs mainly comprised NSPCs and neurons (46.5% βIII‐tubulin⁺ cells). Gamma‐aminobutyric acid (GABA)ergic and dopaminergic/noradrenergic neurons were also observed, along with a network of synaptic proteins. The ES‐NSPCs expressed matriptase and secreted MMP‐2/9, with MMP‐2 activity increasing along time. Fibronectin, laminin and collagen type IV deposition was also detected. Fibrin gels prepared with higher fibrinogen concentrations (8/10 mg/ml) were less permissive to neurite extension and neuronal differentiation, possibly owing to their smaller pore area and higher rigidity. Overall, it is shown that ES‐NSPCs within fibrin are able to establish neuronal networks and to remodel fibrin through MMP secretion and extracellular matrix (ECM) deposition. This three‐dimensional (3D) culture system was also shown to support cell viability, neuronal differentiation and ECM deposition of human ES‐NSPCs. The settled 3D platform is expected to constitute a valuable tool to develop fibrin‐based hydrogels for ES‐NSPC delivery into the injured central nervous system. Copyright © 2016 John Wiley & Sons, Ltd.     

Plasma levels of matrix metalloproteinase‐8 in patients with carotid atherosclerosis

Matrix metalloproteinases (MMPs) are involved in the remodeling of the extracellular matrix (ECM) in the arterial wall during atherogenesis. Collagens are the most abundant proteins in the ECM. MMP‐8 is expressed by cells associated with the development of the atherosclerotic plaque. It cleaves collagen type I three times more potently than two other interstitial collagenases MMP‐1 and MMP‐13. The aim of this study was to investigate whether plasma MMP‐8 values are associated with occurrence of carotid plaque (CP) and possible correlations with clinical and biochemical parameters in carotid atherosclerosis (CA) patients. Total plasma MMP‐8 levels were quantified by ELISA in 63 patients with ultrasonographic evidence of CP presence and 12 controls. Plasma MMP‐8 values were significantly higher in patients with CA compared with controls (median 23.36 ng/ml vs. 13.02 ng/ml, P<0.001) but they did not differ significantly according to gender, smoking and hypertensive status, associated diseases, and use of statins. Statistically significant positive correlations were observed between MMP‐8 plasma values and C reactive protein (r=0.41, P=0.001), urea (r=0.50, P<0.001), aspartate transaminase (r=0.48, P=0.001), and creatinine levels (r=0.38, P=0.006). These results suggest association of MMP‐8 plasma levels with occurrence of CP and correlation with certain biochemical markers. J. Clin. Lab. Anal. 24:246–251, 2010. © 2010 Wiley‐Liss, Inc.     

MMP-2 regulates human platelet activation by interacting with integrin αIIbβ3

Summary. Background: Human platelets contain matrix metalloproteinases (MMPs) that are secreted during platelet activation. Platelet MMPs have been implicated in the regulation of cellular activation and aggregation. Although the proaggregatory effect of MMP‐2 has been demonstrated, the functional mechanism is not clearly understood. Objectives: This work was carried out in order to elucidate the biochemical mechanism of MMP‐2‐associated platelet activation and aggregation. Methods: MMP‐2 binding to the platelet surface was analyzed by flow cytometry. The cell surface target of MMP‐2 was identified in thrombin receptor‐activating peptide‐stimulated platelets by immunoprecipitation, Western blotting and fluorescence microscopy. A recombinant hemopexin‐like domain was used to characterize the nature of MMP‐2 binding to the platelet surface. The functional significance of MMP‐2 in platelet activation was investigated by quantitative measurements of the activation markers P‐selectin (CD62P) and active α_IIbβ₃. The role of MMP‐2 in platelet aggregation was analyzed with an aggregometer. Results: ProMMP‐2 binds to integrin α_IIbβ₃ in stimulated platelets in which proMMP‐2 is converted into MMP‐2. Fibrinogen was able to replace the α_IIbβ₃‐bound MMP‐2. The molecular interaction of MMP‐2 and integrin α_IIbβ₃ was abrogated by the recombinant human hemopexin‐like domain of MMP‐2, leading to reduced cell surface expression of activation markers CD62P and active α_IIbβ₃, and resulting in suppressed platelet aggregation. Conclusion: This work clearly demonstrates that platelet activation and aggregation is regulated by MMP‐2 that specifically interacts with integrin α_IIbβ₃. The C‐terminal hemopexin‐like domain of MMP‐2 is an essential element for binding to α_IIbβ₃.     

Fast clearing RGD‐based near‐infrared fluorescent probes for in vivo tumor diagnosis

A fast clearing hydrophilic near‐infrared (NIR) dye ICG‐Der‐02 was used to constitute tumor targeting contrast agents. Cell adhesion molecule integrin α_vβ₃ served as the target receptor because of its unique expression on almost all sprouting tumor vasculatures. The purpose of this study was to synthesize and compare the properties of integrin α_vβ₃‐targeted, fast clearing NIR probes both in vitro and in vivo for tumor diagnosis. ICG‐Der‐02 was covalently conjugated to three kinds of RGD peptide including linear, monoeric cyclic and dimeric RGD to form three RGD‐based NIR probes. The integrin receptor specificities of these probes were evaluated in vitro by confocal microscopy. The dynamic bio‐distribution and elimination ratse were in vivo real‐time monitored by a near‐infrared imaging system in normal mice. Further, the in vivo tumor targeting abilities of the RGD‐based NIR probes were compared in α_vβ₃‐positive MDA‐MB‐231, U87MG and α_vβ₃‐negtive MCF‐7 xenograft mice models. Three RGD‐based NIR probes were successfully synthesized with good optical properties. In vitro cellular experiments indicated that the probes have a clear binding affinity to α_υβ₃‐positive tumor cells, with a cyclic dimeric RGD probe owing the highest integrin affinity. Dynamic bio‐distributions of these probes showed a rapid clearing rate through the renal pathway. In vivo tumor targeting ability of the RGD‐based porbes was demonstrated on MDA‐MB‐231 and U87MG tumor models. As expected, the c(RGDyK)₂‐ICG‐Der‐02 probe displayed the highest tumor‐to‐normal tissue contrast. The in vitro and in vivo block experiments confirmed the receptor binding specificity of the probes. The hydrophilic dye‐labeled NIR probes exhibited a fast clearing rate and deep tissue penetration capability. Further, the α_υβ₃ receptor affinity of the three RGD‐based NIR probes followed the order of dimer cyclic > monomer cyclic > linear. The results demonstrate potent fast clearing probes for in vivo early tumor diagnosis. Copyright © 2012 John Wiley & Sons, Ltd.     

Beneficial role of l‐arginine in cardiac matrix remodelling in insulin resistant rats

Background The study was performed to determine whether sucrose‐induced insulin resistance could increase the expression of cardiac matrix metalloproteinases (MMPs), indices of matrix remodelling, and whether the addition of 1·25 g die^?1 of L‐arginine (ARG) to a sucrose diet could prevent both the sucrose‐induced metabolic abnormalities and elevated cardiac expression of matrix metalloproteinases in an insulin resistant stage that precedes frank type 2 diabetes. Materials and methods Experiments were performed on 38 male Sprague‐Dawley rats, 16 rats maintained a standard chow diet (ST), 12 rats were switched to a sucrose enriched diet (SU) and 10 rats to a sucrose plus L‐arginine (1·25 g die^?1) enriched diet (SU + ARG) for a period of 8 weeks. After 8 weeks of different diets, an intravenous glucose tolerance test (IVGTT) was performed and samples were drawn for the measurements of insulin, glucose, triglycerides, free fatty acids (FFA), plasma cyclic guanosine‐monophosphate (c‐GMP) and retroperitoneal, omental, epididymal fat pad and heart were dissected and weighed. Results At the end of the study, retroperitoneal fat, heart weight/body weight ratio, fasting plasma glucose, serum insulin, and serum triglyceride levels and integrated insulin area after IVGTT were significantly higher in SU than in SU + ARG and ST. All these parameters were comparable between SU + ARG and ST animals. FFA levels were significantly different among groups, with highest levels in SU and lowest levels in ST. Fasting plasma c‐GMP levels and the integrated c‐GMP area after IVGTT, an index of nitric oxide activity, were significantly lower in SU than in SU + ARG and ST, the result was similar in SU + ARG and in ST MMP‐9 protein expression increased 10·5‐fold, MMP‐2 protein expression increased 2·4‐fold and the expression of tissue inhibitors of metalloproteinase (TIMP‐1) increased 1·7‐fold in SU rats as compared to ST animals. This was accompanied with a significant increase of cardiac triglyceride concentrations. In contrast, cardiac MMP‐9, MMP‐2, and TIMP‐1 protein expressions were not different between SU + ARG and ST animals. Cardiac triglyceride levels were not significantly different between SU + ARG and ST rats. Conclusions SU rats developed insulin resistance and hyperlipidaemia, accompanied with increased fat deposition in the heart and enhanced MMP protein expression. Conversely, ARG supplementation prevents these metabolic abnormalities and restored MMP/TIMP‐1 balance.