Bryan Edward Jones

Soluble Thrombomodulin Protects Ischemic Kidneys

Altered coagulation and inflammation contribute to the pathogenesis of ischemic renal injury. Thrombomodulin is a necessary factor in the anticoagulant protein C pathway and has inherent anti-inflammatory properties. We studied the effect of soluble thrombomodulin (sTM) in a hypoperfusion model of ischemic kidney injury. To markedly reduce infrarenal aortic blood flow and femoral arterial pressures, we clamped the suprarenal aorta of rats, occluding them 90%, for 60 min. Reversible acute kidney injury (AKI) occurred at 24 h in rats subjected to hypoperfusion. Histologic analysis at 24 h revealed acute tubular necrosis (ATN), and intravital two-photon microscopy showed flow abnormalities in the microvasculature and defects of endothelial permeability. Pretreatment with rat sTM markedly reduced both I-R-induced renal dysfunction and tubular histologic injury scores. sTM also significantly improved microvascular erythrocyte flow rates, reduced microvascular endothelial leukocyte rolling and attachment, and minimized endothelial permeability to infused fluorescence dextrans, assessed by intravital quantitative multiphoton microscopy. Furthermore, sTM administered 2 h after reperfusion protected against ischemia-induced renal dysfunction at 24 h and improved survival. By using an sTM variant, we also determined that the protective effects of sTM were independent of its ability to generate activated protein C. These data suggest that sTM may have therapeutic potential for ischemic AKI.

Engineering the proteolytic specificity of activated protein C improves its pharmacological properties

Human activated protein C (APC) is an antithrombotic, antiinflammatory serine protease that plays a central role in vascular homeostasis, and activated recombinant protein C, drotrecogin alfa (activated), has been shown to reduce mortality in patients with severe sepsis. Similar to other serine proteases, functional APC levels are regulated by the serine protease inhibitor family of proteins including α1-antitrypsin and protein C inhibitor. Using APC–substrate modeling, we designed and produced a number of derivatives with the goal of altering the proteolytic specificity of APC such that the variants exhibited resistance to inactivation by protein C inhibitor and α1-antitrypsin yet maintained their primary anticoagulant activity. Substitutions at Leu-194 were of particular interest, because they exhibited 4- to 6-fold reductions in the rate of inactivation in human plasma and substantially increased pharmacokinetic profiles compared with wild-type APC. This was achieved with minimal impairment of the anticoagulant/antithrombotic activity of APC. These data demonstrate the ability to selectively modulate substrate specificity and subsequently affect in vivo performance and suggest therapeutic opportunities for the use of protein C derivatives in disease states with elevated serine protease inhibitor levels.

Distinct Functions of Activated Protein C Differentially Attenuate Acute Kidney Injury

Administration of activated protein C (APC) protects from renal dysfunction, but the underlying mechanism is unknown. APC exerts both antithrombotic and cytoprotective properties, the latter via modulation of protease-activated receptor-1 (PAR-1) signaling. We generated APC variants to study the relative importance of the two functions of APC in a model of LPS-induced renal microvascular dysfunction. Compared with wild-type APC, the K193E variant exhibited impaired anticoagulant activity but retained the ability to mediate PAR-1-dependent signaling. In contrast, the L8W variant retained anticoagulant activity but lost its ability to modulate PAR-1. By administering wild-type APC or these mutants in a rat model of LPS-induced injury, we found that the PAR-1 agonism, but not the anticoagulant function of APC, reversed LPS-induced systemic hypotension. In contrast, both functions of APC played a role in reversing LPS-induced decreases in renal blood flow and volume, although the effects on PAR-1-dependent signaling were more potent. Regarding potential mechanisms for these findings, APC-mediated PAR-1 agonism suppressed LPS-induced increases in the vasoactive peptide adrenomedullin and infiltration of iNOS-positive leukocytes into renal tissue. However, the anticoagulant function of APC was responsible for suppressing LPS-induced stimulation of the proinflammatory mediators ACE-1, IL-6, and IL-18, perhaps accounting for its ability to modulate renal hemodynamics. Both variants reduced active caspase-3 and abrogated LPS-induced renal dysfunction and pathology. We conclude that although PAR-1 agonism is solely responsible for APC-mediated improvement in systemic hemodynamics, both functions of APC play distinct roles in attenuating the response to injury in the kidney.

An Engineered Human Follistatin Variant: Insights into the Pharmacokinetic and Pharmocodynamic Relationships of a Novel Molecule with Broad Therapeutic Potential

Human follistatin is a regulatory glycoprotein with widespread biologic functions, including antiinflammatory activities, wound-healing properties, and muscle-stimulating effects. The role of follistatin in a wide range of biologic activities shows promise for potential clinical application, which has prompted considerable interest in the investigation of the protein as a potential disease-modifying agent. In spite of this potential, the development of follistatin as a broad use biotherapeutic has been severely hindered by a poor understanding and characterization of its pharmacokinetic/pharmacodynamic (PK/PD) relationships. Therefore, to better define these relationships, we performed in-depth analyses of the PK/PD relationships of native follistatin-315 (FST315). Our data indicate that the intrinsic PK/PD properties of native FST315 are poorly suited for acting as a parentally administered biotherapeutic with broad systemic effects. Here, we leveraged protein engineering to modify the PK characteristics of the native molecule by fusing FST315 to a murine IgG1 Fc and removing the intrinsic heparan sulfate-binding activity of follistatin. The engineered variant molecule had ∼100- and ∼1600-fold improvements in terminal half-life and exposure, respectively. In contrast to the native FST315, the variant showed a robust, dose-dependent pharmacological effect when administered subcutaneously on a weekly basis in mouse models of muscle atrophy and degeneration. These studies highlight the underappreciated and critical relationship between optimizing multiple physical and chemical properties of follistatin on its overall PK/PD profile. Moreover, our findings provide the first documented strategy toward the development of a follistatin therapeutic with potential use in patients affected with skeletal muscle diseases.

Myostatin Neutralization Results in Preservation of Muscle Mass and Strength in Preclinical Models of Tumor Induced Muscle Wasting

Skeletal muscle wasting occurs in a great majority of cancer patients with advanced disease and is associated with a poor prognosis and decreased survival. Myostatin functions as a negative regulator of skeletal muscle mass and has recently become a therapeutic target for reducing the loss of skeletal muscle and strength associated with clinical myopathies. We generated neutralizing antibodies to myostatin to test their potential use as therapeutic agents to attenuate the skeletal muscle wasting due to cancer. We show that our neutralizing antimyostatin antibodies significantly increase body weight, skeletal muscle mass, and strength in non–tumor-bearing mice with a concomitant increase in mean myofiber area. The administration of these neutralizing antibodies in two preclinical models of cancer-induced muscle wasting (C26 colon adenocarcinoma and PC3 prostate carcinoma) resulted in a significant attenuation of the loss of muscle mass and strength with no effect on tumor growth. We also show that the skeletal muscle mass– and strength-preserving effect of the antibodies is not affected by the coadministration of gemcitabine, a common chemotherapeutic agent, in both non–tumor-bearing mice and mice bearing C26 tumors. In addition, we show that myostatin neutralization with these antibodies results in the preservation of skeletal muscle mass following reduced caloric intake, a common comorbidity associated with advanced cancer. Our findings support the use of neutralizing antimyostatin antibodies as potential therapeutics for cancer-induced muscle wasting. Mol Cancer Ther; 14(7); 1661–70. ©2015 AACR.