David C. Sane

Calcification after myocardial infarction is independent of amniotic fluid stem cell injection

Ischemic heart disease remains one of the most common causes of mortality in developed countries. Recently, stem cell therapy is being considered for treating ischemic heart diseases. On the other hand, there has been evidence of chondro-osteogenic mass formation after stem cell injection in the heart. In a recent publication, Chiavegato et al. (J Mol Cell Cardiol. 42 (2007) 746-759) has suggested that amniotic fluid-derived stem (AFS) cells cause chondro-osteogenic masses in the infarcted heart. The goal of the current study was to further examine the formation of such masses, specifically, the role of AFS cells in this process. Our results confirm the presence of similar bone-like masses in the left ventricular wall of infarcted rats; however, this phenomenon occurred independent of AFS cell injection into the myocardium. Moreover, AFS cell injection did not increase the presence of chondro-osteogenic masses. Echocardiographic analysis of large infarctions in rats frequently revealed the presence of echogenic structures in the left ventricular wall. We further demonstrated a significant relationship between the infarction size and chondro-osteogenic formation and subsequent decrease in cardiac function. Collectively, our study indicates that chondro-osteogenic differentiation can take place in infarcted rat heart independent of cell injection. These results have significant implications for future design and testing of stem cell therapy for treatment of cardiac muscle diseases.

N -Glycosylation engineering of tobacco plants to produce asialoerythropoietin

Erythropoietin (EPO) is a glycoprotein hormone that displays both hematopoietic and tissue-protective functions by binding to two distinct receptors. Recombinant human EPO (rhuEPO) is widely used for the treatment of anemia, but its use for tissue protection is limited because of potentially harmful increases in red blood cell mass when higher doses of rhuEPO are used. Recent studies have shown that asialoerythropoietin (asialo-rhuEPO), a desialylated form of rhuEPO, lacks hematopoietic activity, but retains cytoprotective activity. Currently, a small amount of asialo-rhuEPO is produced by enzymatic desialylation of rhuEPO. The prohibitive cost of rhuEPO, however, is a major limitation of this method. Plants have the ability to synthesize complex N-glycans, but lack enzymatic activities to add sialic acid and β1,4-galactose to N-glycan chains. Plants could be genetically engineered to produce asialo-rhuEPO by introducing human β1,4-galactosyltransferase. The penultimate β1,4-linked galactose residues are important for in vivo biological activity. In this proof of concept study, we show that tobacco plants co-expressing human β1,4-galactosyltransferase and EPO genes accumulated asialo-rhuEPO. Purified asialo-rhuEPO binds to an Erythrina cristagalli lectin column, indicating that its N-glycan chains bear terminal β1,4-galactose residues and that the co-expressed GalT is functionally active. Asialo-rhuEPO interacted with the EPO receptor (EPOR) with similar affinity as rhuEPO, implying that it was properly folded. The strategy described here provides a straightforward way to produce asialo-rhuEPO for research and therapeutic purposes.Key messageN-glycosylation pathway in tobacco plants could be genetically engineered to produce a tissue-protective cytokine, asialoerythropoietin (a desialylated form of human hormone erythropoietin).

Weekly doxorubicin increases coronary arteriolar wall and adventitial thickness.

Background Doxorubicin (DOX) is associated with premature cardiovascular events including myocardial infarction. This study was performed to determine if the weekly administration of DOX influenced coronary arteriolar medial and/or adventitial wall thickening. Methods Thirty-two male Sprague-Dawley rats aged 25.1± 2.4 weeks were randomly divided into three groups and received weekly intraperitoneal injections of normal saline (saline, nu200a=u200a7), or low (1.5 mg/kg to 1.75 mg/kg, nu200a=u200a14) or high (2.5 mg/kg, nu200a=u200a11) doses of DOX. The animals were treated for 2–12 weeks, and euthanized at pre-specified intervals (2, 4, 7, or 10+ weeks) to obtain histopathologic assessments of coronary arteriolar lumen diameter, medial wall thickness, adventitial wall thickness, and total wall thickness (medial thickness + adventitial thickness). Results Lumen diameter was similar across all groups (saline: 315±34 µm, low DOX: 286±24 µm, high DOX: 242±27 µm; pu200a=u200a0.22). In comparison to animals receiving weekly saline, animals receiving weekly injections of 2.5 mg/kg of DOX experienced an increase in medial (23±2µm vs. 13±3µm; pu200a=u200a0.005), and total wall thickness (51±4µm vs. 36±5µm; pu200a=u200a0.022), respectively. These increases, as well as adventitial thickening became more prominent after normalizing for lumen diameter (p<0.05 to p<0.001) and after adjusting for age, weight, and total cumulative DOX dose (pu200a=u200a0.02 to pu200a=u200a0.01). Animals receiving low dose DOX trended toward increases in adventitial and total wall thickness after normalization to lumen diameter and accounting for age, weight, and total cumulative DOX dose (pu200a=u200a0.06 and 0.09, respectively). Conclusion In conclusion, these data demonstrate that weekly treatment of rats with higher doses of DOX increases coronary arteriolar medial, adventitial, and total wall thickness. Future studies are warranted to determine if DOX related coronary arteriolar effects are reversible or preventable, exacerbate the known cardiomyopathic effects of DOX, influence altered resting or stress-induced myocardial perfusion, or contribute to the occurrence of myocardial infarction.

Cytoprotective Effect of Recombinant Human Erythropoietin Produced in Transgenic Tobacco Plants

Asialo-erythropoietin, a desialylated form of human erythropoietin (EPO) lacking hematopoietic activity, is receiving increased attention because of its broader protective effects in preclinical models of tissue injury. However, attempts to translate its protective effects into clinical practice is hampered by unavailability of suitable expression system and its costly and limit production from expensive mammalian cell-made EPO (rhuEPOM) by enzymatic desialylation. In the current study, we took advantage of a plant-based expression system lacking sialylating capacity but possessing an ability to synthesize complex N-glycans to produce cytoprotective recombinant human asialo-rhuEPO. Transgenic tobacco plants expressing asialo-rhuEPO were generated by stably co-expressing human EPO and β1,4-galactosyltransferase (GalT) genes under the control of double CaMV 35S and glyceraldehyde-3-phosphate gene (GapC) promoters, respectively. Plant-produced asialo-rhuEPO (asialo-rhuEPOP) was purified by immunoaffinity chromatography. Detailed N-glycan analysis using NSI-FTMS and MS/MS revealed that asialo-rhuEPOP bears paucimannosidic, high mannose-type and complex N-glycans. In vitro cytoprotection assays showed that the asialo-rhuEPOP (20 U/ml) provides 2-fold better cytoprotection (44%) to neuronal-like mouse neuroblastoma cells from staurosporine-induced cell death than rhuEPOM (21%). The cytoprotective effect of the asialo-rhuEPOP was found to be mediated by receptor-initiated phosphorylation of Janus kinase 2 (JAK2) and suppression of caspase 3 activation. Altogether, these findings demonstrate that plants are a suitable host for producing cytoprotective rhuEPO derivative. In addition, the general advantages of plant-based expression system can be exploited to address the cost and scalability issues related to its production.

Effects of Vessel Tortuosity on Coronary Hemodynamics: An Idealized and Patient-Specific Computational Study.

Although coronary tortuosity can influence the hemodynamics of coronary arteries, the relationship between tortuosity and flow has not been thoroughly investigated partly due to the absence of a widely accepted definition of tortuosity and the lack of patient-specific studies that analyze complete coronary trees. Using a computational approach we investigated the effects of tortuosity on coronary flow parameters including pressure drop, wall shear stress, and helical flow strength as measured by helicity intensity. Our analysis considered idealized and patient-specific geometries. Overall results indicate that perfusion pressure decreases with increased tortuosity, but the patient-specific results show that more tortuous vessels have higher physiological wall shear stress values. Differences between the idealized and patient-specific results reveal that an accurate representation of coronary tortuosity must account for all relevant geometric aspects, including curvature imposed by the heart shape. The patient-specific results exhibit a strong correlation between tortuosity and helicity intensity, and the corresponding helical flow contributes directly to the observed increase in wall shear stress. Therefore, helicity intensity may prove helpful in developing a universal parameter to describe tortuosity and assess its impact on patient health. Our data suggest that increased tortuosity could have a deleterious impact via a reduction in coronary perfusion pressure, but the attendant increase in wall shear stress could afford protection against atherosclerosis.

Two-step purification procedure for recombinant human asialoerythropoietin expressed in transgenic plants

Asialoerythropoietin (asialo-EPO) is a desialylated form of human glycoprotein hormone erythropoietin (EPO), which has been reported to be neuro-, cardio-, and renoprotective in animal models of organ injuries. Since the current method of production of asialo-EPO from mammalian cell-made recombinant human EPO (rhuEPO(M)) by enzymatic desialylation is not commercially viable, we and others used plant-based expression systems to produce recombinant human asialo-EPO (asialo-rhuEPO(P)). Despite achieving high expression levels in plants, its purification from plant extracts has remained a greater challenge, which has prevented studying its tissue-protective effects and translating it into clinical practice. In this study, a procedure was developed to purify asialo-rhuEPO(P) from transgenic tobacco leaf tissues in two steps: ion-exchange chromatography based on its high pI (8.75) to separate it from acidic plant proteins, and immunoaffinity chromatography to obtain pure asialo-rhuEPO(P). Using this process, up to 31% of the asialo-rhuEPO(P) could be recovered to near homogeneity from plant extracts. This work demonstrates that asialo-rhuEPO(P) expressed in tobacco plants could be purified in high yield and purity using minimal steps, which might be suitable for scale-up. Furthermore, the ion-exchange chromatography step together with the use of protein-specific antibody column could be used to purify a wide variety of basic recombinant proteins from transgenic leaf tissues.

Cardiac manifestations of cutaneous disorders.

A number of cutaneous disorders encountered by the dermatologist have overlapping cardiac pathology. In recent years, many genetic linkages common to pathological processes in the cutaneous and cardiovascular systems have been identified. This review will describe primary cutaneous disorders with potential cardiac manifestations, including congenital syndromes, inherited cutaneous disorders associated with later cardiovascular disease, and syndromes associated with early cardiovascular pathology. The dermatologist may be the first to diagnose cutaneous findings associated with underlying cardiovascular disease; therefore, it is of prime importance for the dermatologist to be aware of these associations and to direct the appropriate workup.

Plant-Produced Asialo-Erythropoietin Restores Pancreatic Beta-Cell Function by Suppressing Mammalian Sterile-20-like Kinase (MST1) and Caspase-3 Activation

Pancreatic beta-cell death adversely contributes to the progression of both type I and II diabetes by undermining beta-cell mass and subsequently diminishing endogenous insulin production. Therapeutics to impede or even reverse the apoptosis and dysfunction of beta-cells are urgently needed. Asialo-rhuEPO, an enzymatically desialylated form of recombinant human erythropoietin (rhuEPO), has been shown to have cardioprotective and neuroprotective functions but with no adverse effects like that of sialylated rhuEPO. Heretofore, the anti-apoptotic effect of asialo-rhuEPO on pancreatic beta-cells has not been reported. In the current study, we investigated the cytoprotective properties of plant-produced asialo-rhuEPO (asialo-rhuEPOP) against staurosporine-induced cell death in the pancreatic beta-cell line RIN-m5F. Our results showed that 60 IU/ml asialo-rhuEPOP provided 41% cytoprotection while 60 IU/ml rhuEPO yielded no effect. Western blotting results showed that asialo-rhuEPOP treatment inhibited both MST1 and caspase-3 activation with the retention of PDX1 and insulin levels close to untreated control cells. Our study provides the first evidence indicating that asialo-rhuEPOP-mediated protection involves the reduction of MST1 activation, which is considered a key mediator of apoptotic signaling in beta-cells. Considering the many advantages its plant-based expression, asialo-rhuEPOP could be potentially developed as a novel and inexpensive agent to treat or prevent diabetes after further performing studies in cell-based and animal models of diabetes.

C-Terminally fused affinity Strep-tag II is removed by proteolysis from recombinant human erythropoietin expressed in transgenic tobacco plants

Key messageC-terminally fusedStrep-tag II is removed from rhuEPO expressed in tobacco plants. The finding suggests that direct fusion of purification tags at theC-terminus of rhuEPO should be avoided.AbstractAsialo-erythropoietin (asialo-EPO), a desialylated form of EPO, is a potent tissue-protective agent. Recently, we and others have exploited a low-cost plant-based expression system to produce recombinant human asialo-EPO (asialo-rhuEPOP). To facilitate purification from plant extracts, Strep-tag II was engineered at the C-terminus of EPO. Although asialo-rhuEPOP was efficiently expressed in transgenic tobacco plants, affinity purification based on Strep-tag II did not result in the recovery of the protein. In this study, we investigated the stability of Strep-tag II tagged asialo-rhuEPOP expressed in tobacco plants to understand whether this fused tag is cleaved or inaccessible. Sequencing RT-PCR products confirmed that fused DNA sequences encoding Strep-tag II were properly transcribed, and three-dimensional protein structure model revealed that the tag must be fully accessible. However, Western blot analysis of leaf extracts and purified asialo-rhuEPOP revealed that the Strep-tag II was absent on the protein. Additionally, no peptide fragment containing Strep-tag II was identified in the LC–MS/MS analysis of purified protein further supporting that the affinity tag was absent on asialo-rhuEPOP. However, Strep-tag II was detected on asialo-rhuEPOP that was retained in the endoplasmic reticulum, suggesting that the Strep-tag II is removed during protein secretion or extraction. These findings together with recent reports that C-terminally fused Strep-tag II or IgG Fc domain are also removed from EPO in tobacco plants, suggest that its C-terminus may be highly susceptible to proteolysis in tobacco plants. Therefore, direct fusion of purification tags at the C-terminus of EPO should be avoided while expressing it in tobacco plants.

Interference of thrombin in immunological assays for hirudin specific antibodies.

Recombinant hirudins (desirudin, lepirudin) are direct thrombin inhibitors administered as anticoagulants for heparin-induced thrombocytopenia (HIT) and venous thromboembolism (VTE) prophylaxis. Although these small polypeptides are widely used, concern exists over reports of antigenicity. In the largest study of r-hirudin immunogenicity to-date, we evaluated the prevalence, quantity and specificity of IgG immune responses to desirudin (15 mg SC q12h for as long as clinically required) in 245 surgical and medically-ill subjects enrolled in DESIRABLE, a multicenter, open-label, clinical trial of hospitalized patients requiring VTE prophylaxis. Sera obtained before and 30 days after desirudin administration were analyzed for IgG anti-desirudin by immunoenzymetric assay using immobilized desirudin to bind desirudin-reactive antibody and peroxidase conjugated monoclonal-anti-human IgG Fc to detect bound IgG antibody. Of 245 study subjects, 19 (7.7%) were antibody responders (>2-fold increase in IgG antibody levels with >50% inhibition by desirudin 30 days post-treatment). There were no differences between responders and non-responders in incidence of clinical outcomes or bleeding-related adverse events. Forty-six patients had detectable desirudin-reactive IgG antibody prior to treatment, with no significant increase in antibody levels after exposure and no increase in clinical events. The origin of pre-existing hirudin-reactive IgG antibody requires further investigation involving suspected anti-thrombin-thrombin interactions. These results indicate a low potential for immunogenicity, with <8% of patients developing IgG antibodies after desirudin administration for VTE prophylaxis. In contrast to reports on lepirudin, production of anti-hirudin antibodies to desirudin has no apparent effect on clinical events.