Sergei Merkulov

Differential proteomics analysis of the surface heterogeneity of dextran iron oxide nanoparticles and the implications for their in vivo clearance

In order to understand the role of plasma proteins in the rapid liver clearance of dextran-coated superparamagnetic iron oxide (SPIO) in vivo, we analyzed the full repertoire of SPIO-binding blood proteins using novel two-dimensional differential mass spectrometry approach. The identified proteins showed specificity for surface domains of the nanoparticles: mannan-binding lectins bound to the dextran coating, histidine-rich glycoprotein and kininogen bound to the iron oxide part, and the complement lectin and contact clotting factors were secondary binders. Nanoparticle clearance studies in knockout mice suggested that these proteins, as well as several previously identified opsonins, do not play a significant role in the SPIO clearance. However, both the dextran coat and the iron oxide core remained accessible to specific probes after incubation of SPIO in plasma, suggesting that the nanoparticle surface could be available for recognition by macrophages, regardless of protein coating. These data provide guidance to rational design of bioinert, long-circulating nanoparticles.

Recognition of dextran-superparamagnetic iron oxide nanoparticle conjugates (Feridex) via macrophage scavenger receptor charged domains.

Dextran-coated superparamagnetic iron oxide nanoparticles (dextran-SPIO conjugates) offer the attractive possibility of enhancing MRI imaging sensitivity so that small or diffuse lesions can be detected. However, systemically injected SPIOs are rapidly removed by macrophages. We engineered embryonic cells (HEK293T) to express major macrophage scavenger receptor (SR) subtypes including SR-AI, MARCO, and endothelial receptor collectin-12. These SRs possess a positively charged collagen-like (CL) domain and they promoted SPIO uptake, while the charge neutral lipoprotein receptor SR-BI did not. In silico modeling indicated a positive net charge on the CL domain and a net negative charge on the cysteine-rich (CR) domain of MARCO and SR-AI. In vitro experiments revealed that CR domain deletion in SR-AI boosted uptake of SPIO 3-fold, while deletion of MARCOs CR domain abolished this uptake. These data suggest that future studies might productively focus on the validation and further exploration of SR charge fields in SPIO recognition.

Prolylcarboxypeptidase promotes angiogenesis and vascular repair

Prolylcarboxypeptidase (PRCP) is associated with leanness, hypertension, and thrombosis. PRCP-depleted mice have injured vessels with reduced Kruppel-like factor (KLF)2, KLF4, endothelial nitric oxide synthase (eNOS), and thrombomodulin. Does PRCP influence vessel growth, angiogenesis, and injury repair? PRCP depletion reduced endothelial cell growth, whereas transfection of hPRCP cDNA enhanced cell proliferation. Transfection of hPRCP cDNA, or an active site mutant (hPRCPmut) rescued reduced cell growth after PRCP siRNA knockdown. PRCP-depleted cells migrated less on scratch assay and murine PRCP(gt/gt) aortic segments had reduced sprouting. Matrigel plugs in PRCP(gt/gt) mice had reduced hemoglobin content and angiogenic capillaries by platelet endothelial cell adhesion molecule (PECAM) and NG2 immunohistochemistry. Skin wounds on PRCP(gt/gt) mice had delayed closure and reepithelialization with reduced PECAM staining, but increased macrophage infiltration. After limb ischemia, PRCP(gt/gt) mice also had reduced reperfusion of the femoral artery and angiogenesis. On femoral artery wire injury, PRCP(gt/gt) mice had increased neointimal formation, CD45 staining, and Ki-67 expression. Alternatively, combined PRCP(gt/gt) and MRP-14(-/-) mice were protected from wire injury with less neointimal thickening, leukocyte infiltration, and cellular proliferation. PRCP regulates cell growth, angiogenesis, and the response to vascular injury. Combined with its known roles in blood pressure and thrombosis control, PRCP is positioned as a key regulator of vascular homeostasis.

Contact activation of kallikrein-kinin system by superparamagnetic iron oxide nanoparticles in vitro and in vivo.

Previously we reported that plasma kallikrein and high molecular weight kininogen attach to the surface of dextran-coated superparamagnetic iron oxide nanoparticles (SPION) through the incompletely covered iron oxide core (Simberg et al., Biomaterials, 2009). Here we show that SPION also activate kallikrein-kinin system in vitro and in vivo. The serine protease activity of kallikrein was stably associated with SPION and could be detected on the nanoparticles even after extensive washing steps. The enzymatic activity was not detectable in kininogen-deficient and Factor XII-deficient plasma. The enzymatic activation could be blocked by precoating SPION with histidine-rich Domain 5 (D5) of kininogen. Importantly, the kallikrein activity was detectable in plasma of SPION-injected, but not of D5/SPION-injected mice. Tumor-targeted SPION when injected into kininogen-deficient and control mice, produced high levels of vascular clotting in tumors, suggesting that kallikrein activation is not responsible for the nanoparticle-induced thrombosis. These data could help in understanding the toxicity of nanomaterials and could be used in designing nanoparticles with controlled enzymatic activity.

In Vivo Cardiac Myosin Binding Protein C Gene Transfer Rescues Myofilament Contractile Dysfunction in Cardiac Myosin Binding Protein C Null Mice

Background —Decreased expression of cardiac myosin binding protein C (cMyBPC) in the heart has been implicated as a consequence of mutations in cMyBPC that lead to abnormal contractile function at the myofilament level, thereby contributing to the development of hypertrophic cardiomyopathy in humans. It is has not been established whether increasing the levels of cMyBPC in the intact heart can improve myofilament and in vivo contractile function and attenuate maladaptive remodeling processes due to reduced levels of cMyBPC. Methods and Results —We performed in vivo gene transfer of cMyBPC by direct injection into the myocardium of cMyBPC deficient (cMyBPC-/-) mice, and mechanical experiments were conducted on skinned myocardium isolated from cMyBPC-/- hearts 21 days and 20 weeks following gene transfer. Cross-bridge kinetics in skinned myocardium isolated from cMyBPC-/- hearts following cMyBPC gene transfer were significantly slowed compared to untreated cMyBPC-/- myocardium, and were comparable to wild-type (WT) myocardium, and cMyBPC-/- myocardium that was reconstituted with recombinant cMyBPC in vitro . cMyBPC content in cMyBPC-/- skinned myocardium following in vivo cMyBPC gene transfer or in vitro cMyBPC reconstitution was similar to WT levels. In vivo echocardiography studies of cMyBPC-/- hearts following cMyBPC gene transfer revealed improved systolic and diastolic contractile function and reductions in LV wall thickness. Conclusions —This proof-of-concept study demonstrates that gene therapy designed to increase expression of cMyBPC in the cMyBPC deficient myocardium can improve myofilament and in vivo contractile function, suggesting that cMyBPC gene therapy may be a viable approach for treatment cardiomyopathies due to mutations in cMyBPC.Background—Decreased expression of cardiac myosin binding protein C (cMyBPC) in the heart has been implicated as a consequence of mutations in cMyBPC that lead to abnormal contractile function at the myofilament level, thereby contributing to the development of hypertrophic cardiomyopathy in humans. It has not been established whether increasing the levels of cMyBPC in the intact heart can improve myofilament and in vivo contractile function and attenuate maladaptive remodeling processes because of reduced levels of cMyBPC. Methods and Results—We performed in vivo gene transfer of cMyBPC by direct injection into the myocardium of cMyBPC-deficient (cMyBPC−/−) mice, and mechanical experiments were conducted on skinned myocardium isolated from cMyBPC−/− hearts 21 days and 20 weeks after gene transfer. Cross-bridge kinetics in skinned myocardium isolated from cMyBPC−/− hearts after cMyBPC gene transfer were significantly slower compared with untreated cMyBPC−/− myocardium and were comparable to wild-type myocardium and cMyBPC−/− myocardium that was reconstituted with recombinant cMyBPC in vitro. cMyBPC content in cMyBPC−/− skinned myocardium after in vivo cMyBPC gene transfer or in vitro cMyBPC reconstitution was similar to wild-type levels. In vivo echocardiography studies of cMyBPC−/− hearts after cMyBPC gene transfer revealed improved systolic and diastolic contractile function and reductions in left ventricular wall thickness. Conclusions—This proof-of-concept study demonstrates that gene therapy designed to increase expression of cMyBPC in the cMyBPC-deficient myocardium can improve myofilament and in vivo contractile function, suggesting that cMyBPC gene therapy may be a viable approach for treatment of cardiomyopathies because of mutations in cMyBPC.

In Vivo cMyBPC Gene Transfer Rescues Myofilament Contractile Dysfunction in cMyBPC Null Mice

Background —Decreased expression of cardiac myosin binding protein C (cMyBPC) in the heart has been implicated as a consequence of mutations in cMyBPC that lead to abnormal contractile function at the myofilament level, thereby contributing to the development of hypertrophic cardiomyopathy in humans. It is has not been established whether increasing the levels of cMyBPC in the intact heart can improve myofilament and in vivo contractile function and attenuate maladaptive remodeling processes due to reduced levels of cMyBPC. Methods and Results —We performed in vivo gene transfer of cMyBPC by direct injection into the myocardium of cMyBPC deficient (cMyBPC-/-) mice, and mechanical experiments were conducted on skinned myocardium isolated from cMyBPC-/- hearts 21 days and 20 weeks following gene transfer. Cross-bridge kinetics in skinned myocardium isolated from cMyBPC-/- hearts following cMyBPC gene transfer were significantly slowed compared to untreated cMyBPC-/- myocardium, and were comparable to wild-type (WT) myocardium, and cMyBPC-/- myocardium that was reconstituted with recombinant cMyBPC in vitro . cMyBPC content in cMyBPC-/- skinned myocardium following in vivo cMyBPC gene transfer or in vitro cMyBPC reconstitution was similar to WT levels. In vivo echocardiography studies of cMyBPC-/- hearts following cMyBPC gene transfer revealed improved systolic and diastolic contractile function and reductions in LV wall thickness. Conclusions —This proof-of-concept study demonstrates that gene therapy designed to increase expression of cMyBPC in the cMyBPC deficient myocardium can improve myofilament and in vivo contractile function, suggesting that cMyBPC gene therapy may be a viable approach for treatment cardiomyopathies due to mutations in cMyBPC.Background—Decreased expression of cardiac myosin binding protein C (cMyBPC) in the heart has been implicated as a consequence of mutations in cMyBPC that lead to abnormal contractile function at the myofilament level, thereby contributing to the development of hypertrophic cardiomyopathy in humans. It has not been established whether increasing the levels of cMyBPC in the intact heart can improve myofilament and in vivo contractile function and attenuate maladaptive remodeling processes because of reduced levels of cMyBPC. Methods and Results—We performed in vivo gene transfer of cMyBPC by direct injection into the myocardium of cMyBPC-deficient (cMyBPC−/−) mice, and mechanical experiments were conducted on skinned myocardium isolated from cMyBPC−/− hearts 21 days and 20 weeks after gene transfer. Cross-bridge kinetics in skinned myocardium isolated from cMyBPC−/− hearts after cMyBPC gene transfer were significantly slower compared with untreated cMyBPC−/− myocardium and were comparable to wild-type myocardium and cMyBPC−/− myocardium that was reconstituted with recombinant cMyBPC in vitro. cMyBPC content in cMyBPC−/− skinned myocardium after in vivo cMyBPC gene transfer or in vitro cMyBPC reconstitution was similar to wild-type levels. In vivo echocardiography studies of cMyBPC−/− hearts after cMyBPC gene transfer revealed improved systolic and diastolic contractile function and reductions in left ventricular wall thickness. Conclusions—This proof-of-concept study demonstrates that gene therapy designed to increase expression of cMyBPC in the cMyBPC-deficient myocardium can improve myofilament and in vivo contractile function, suggesting that cMyBPC gene therapy may be a viable approach for treatment of cardiomyopathies because of mutations in cMyBPC.

Inhibition of angiogenesis by cleaved high molecular weight kininogen (HKa) and HKa domain 5.

High molecular weight kininogen (HK) is an abundant, multi-domain plasma protein that circulates in plasma primarily in its single chain form. Proteolytic cleavage of HK by plasma kallikrein releases the vasoactive nanopeptide bradykinin (BK), and converts HK into two-chain HK (HKa). BK appears to have pro-angiogenic activity, most likely mediated through binding to B1 and B2 receptors on endothelial cells. Conversely, HKa and its domain 5, but not (single chain) HK, have potent anti-angiogenic activity comparable to other endogenous angiogenesis inhibitors. The mechanism by which HKa exerts its anti-angiogenic activity remains controversial, but appears to involve binding to cell surface tropomyosin and induction of apoptosis of proliferating endothelial cells. A role for tropomyosin in mediating the anti-angiogenic signals of other anti-angiogenic proteins such as endostatin and histidine-proline-rich glycoprotein (HPRG) has also been reported. Here we review the physiological importance of high molecular weight kininogen in angiogenesis, with emphasis on the mechanism(s) by which this activity is mediated.

Residues required for phosphorylation of translation initiation factor eIF2α under diverse stress conditions are divergent between yeast and human

PERK, PKR, HRI and GCN2 are the four mammalian kinases that phosphorylate the α subunit of the eukaryotic translation initiation factor 2 (eIF2α) on Ser51. This phosphorylation event is conserved among many species and attenuates protein synthesis in response to diverse stress conditions. In contrast, Saccharmyces cerevisiae expresses only the GCN2 kinase. It was demonstrated previously in S. cerevisiae that single point mutations in eIF2αs N-terminus severely impaired phosphorylation at Ser51. To assess whether similar recognition patterns are present in mammalian eIF2α, we expressed human eIF2αs with these mutations in mouse embryonic fibroblasts and assessed their phosphorylation under diverse stress conditions. Some of the mutations prevented the stress-induced phosphorylation of eIF2α by all mammalian kinases, thus defining amino acid residues in eIF2α (Gly 30, Leu 50, and Asp 83) that are required for substrate recognition. We also identified residues that were less critical or not required for recognition by the mammalian kinases (Ala 31, Met 44, Lys 79, and Tyr 81), even though they were essential for recognition of the yeast eIF2α by GCN2. We propose that mammalian eIF2α kinases evolved to maximize their interactions with the evolutionarily conserved Ser51 residue of eIF2α in response to diverse stress conditions, thus adding to the complex signaling pathways that mammalian cells have over simpler organisms.

Chimeric Glutathione S-Transferases Containing Inserts of Kininogen Peptides POTENTIAL NOVEL PROTEIN THERAPEUTICS

Background: Short human kininogen (HK) peptides can be used as therapeutic agents. Results: The biological activity of the HK peptides is dramatically enhanced following insertion into GST. Conclusion: The efficacy of short peptides can be enhanced by insertion into GST without affecting GST structure. Significance: Intra-backbone insertions of small peptides into easily expressed, well characterized soluble proteins may help to create novel protein therapeutics. The study of synthetic peptides corresponding to discrete regions of proteins has facilitated the understanding of protein structure-activity relationships. Short peptides can also be used as powerful therapeutic agents. However, in many instances, small peptides are prone to rapid degradation or aggregation and may lack the conformation required to mimic the functional motifs of the protein. For peptides to function as pharmacologically active agents, efficient production or expression, high solubility, and retention of biological activity through purification and storage steps are required. We report here the design, expression, and functional analysis of eight engineered GST proteins (denoted GSHKTs) in which peptides ranging in size from 8 to 16 amino acids and derived from human high molecular weight kininogen (HK) domain 5 were inserted into GST (between Gly-49 and Leu-50). Peptides derived from HK are known to inhibit cell proliferation, angiogenesis, and tumor metastasis, and the biological activity of the HK peptides was dramatically (>50-fold) enhanced following insertion into GST. GSHKTs are soluble and easily purified from Escherichia coli by affinity chromatography. Functionally, these hybrid proteins cause inhibition of endothelial cell proliferation. Crystallographic analysis of GSHKT10 and GSHKT13 (harboring 10- and 13-residue HK peptides, respectively) showed that the overall GST structure was not perturbed. These results suggest that the therapeutic efficacy of short peptides can be enhanced by insertion into larger proteins that are easily expressed and purified and that GST may potentially be used as such a carrier.