Brian W. Grinnell

The MAD-Related Protein Smad7 Associates with the TGFβ Receptor and Functions as an Antagonist of TGFβ Signaling

Abstract TGFβ signaling is initiated when the type I receptor phosphorylates the MAD-related protein, Smad2, on C-terminal serine residues. This leads to Smad2 association with Smad4, translocation to the nucleus, and regulation of transcriptional responses. Here we demonstrate that Smad7 is an inhibitor of TGFβ signaling. Smad7 prevents TGFβ-dependent formation of Smad2/Smad4 complexes and inhibits the nuclear accumulation of Smad2. Smad7 interacts stably with the activated TGFβ type I receptor, thereby blocking the association, phosphorylation, and activation of Smad2. Furthermore, mutations in Smad7 that interfere with receptor binding disrupt its inhibitory activity. These studies thus define a novel function for MAD-related proteins as intracellular antagonists of the type I kinase domain of TGFβ family receptors.

Recombinant human activated protein C attenuates the inflammatory response in endothelium and monocytes by modulating nuclear factor-κB

ObjectiveTo review the anti-inflammatory and anti-apoptotic properties of drotrecogin alfa (activated) (recombinant human activated protein C), emphasizing its modulatory effects on endothelial nuclear factor-&kgr;B. We propose a broad anti-inflammatory effect of drotrecogin alfa (activated), acting on both endothelium and monocytes. Data SourcesA selected review of the published literature on nuclear factor-&kgr;B, severe sepsis, and the use of drotrecogin alfa (activated) in clinical and preclinical models, together with data derived from preclinical gene profiling of model systems. Data Extraction and SynthesisData from the PROWESS trial support the preclinical evidence of an antithrombotic effect of drotrecogin alfa (activated). Anti-inflammatory effects through reduction of thrombin generation and through thrombin-independent mechanisms in mononuclear and endothelial cells are reviewed. Inhibition of apoptosis is used as an example of the protective effect of drotrecogin alfa (activated) on endothelial and mononuclear cell dysfunction. ConclusionsDrotrecogin alfa (activated) acts as a modulator of nuclear factor-&kgr;B to aid in the host immune response in endothelium and monocytes. Extrapolation of gene array findings to explain apoptosis in endothelium and monocytes, coupled with emerging preclinical reports, provides evidence to support the role of drotrecogin alfa (activated) in modulating nuclear factor-&kgr;B.

Tubulo-Interstitial Nephritis Associated with Polyomavirus (BK Type) Infection

We studied viral injury to the kidney in a six-year-old boy with hyperimmunoglobulin M immunodeficiency who presented with irreversible acute renal failure and eventually died after five months of dialysis. Renal biopsy at the time of his presentation revealed a predominantly tubulo-interstitial process with numerous viral inclusions that were identified as polyomavirus. Urine cultures showed a massive viruria with BK-type, polyomavirus. The kidney disease was end stage, with persistence of BK virus identified by morphologic techniques and by culture. DNA hybridization analysis showed virus in low concentration in the lymph nodes, spleen, and lungs. The marked viruria, the high concentration of BK virus, and the extensive distribution of viral antigen throughout the kidney all suggest that infection with BK virus was the basis of the severe renal parenchymal injury.

Recombinant human activated protein C: a system modulator of vascular function for treatment of severe sepsis.

ObjectiveTo review the mechanisms of action and rationale for the use of recombinant human activated protein C in the treatment of severe sepsis. Specifically, we focus on the mechanisms of action in the protein C pathway that converge to modulate the pathophysiology of severe inflammatory disease and sepsis. This analysis includes a discussion of the role of activated protein C in directly modulating cell system biology, independent of antithrombotic activity. Data Sources/Study Selection Published research and review articles relating to the protein C pathway, recombinant human protein C, and the role of protein C in sepsis. Data were also derived from broad gene profiling in model systems of endothelial dysfunction. Data Extraction and Synthesis Relevant studies were included to support discussion of the unique mechanistic aspect of protein C and its role in the pathogenesis of severe sepsis. We discuss the potential of activated protein C as a unique system modulator for the treatment of severe sepsis and other systemic inflammatory responses that result in microvascular coagulopathy, endothelial dysfunction, and vascular bed failure. ConclusionsThe protein C pathway plays a unique role in modulating vascular function. As an antithrombotic/profibrinolytic agent, it plays a clear role in maintaining vascular patency. Moreover, it has anti-inflammatory properties and appears to play a unique role as an antiapoptotic and endothelial cell survival factor. In states of systemic inflammatory activation, loss of protein C due to consumptive processes results in a compromised ability to modulate coagulation as well as inflammatory and cell survival functions. This compromise leads to vascular dysfunction, end-organ failure, and death. Replacement with recombinant human activated protein C offers a system-modulating approach to improved outcome.

Crucial role of the protein C pathway in governing microvascular inflammation in inflammatory bowel disease

Endothelial protein C receptor (EPCR) and thrombomodulin (TM) are expressed at high levels in the resting microvasculature and convert protein C (PC) into its activated form, which is a potent anticoagulant and antiinflammatory molecule. Here we provide evidence that in Crohn disease (CD) and ulcerative colitis (UC), the 2 major forms of inflammatory bowel disease (IBD), there was loss of expression of endothelial EPCR and TM, which in turns caused impairment of PC activation by the inflamed mucosal microvasculature. In isolated human intestinal endothelial cells, administration of recombinant activated PC had a potent antiinflammatory effect, as demonstrated by downregulated cytokine-dependent cell adhesion molecule expression and chemokine production as well as inhibited leukocyte adhesion. In vivo, administration of activated PC was therapeutically effective in ameliorating experimental colitis as evidenced by reduced weight loss, disease activity index, and histological colitis scores as well as inhibited leukocyte adhesion to the inflamed intestinal vessels. The results suggest that the PC pathway represents a new system crucially involved in governing intestinal homeostasis mediated by the mucosal microvasculature. Restoring the PC pathway may represent a new therapeutic approach to suppress intestinal inflammation in IBD.

Leukocyte and endothelial cell interactions in sepsis: relevance of the protein C pathway.

Objective:To give an overview of leukocyte and endothelial cell interactions in sepsis and to explore the role of the protein C pathway in modulating the innate immune response via its anti-inflammatory properties. Data Source:Novel in vitro data and a MEDLINE search for the terms “activated protein C,” “recombinant human activated protein C,” “inflammation,” “leukocyte adhesion,” and “sepsis” were used, along with clinical trial databases from the PROWESS trial and a phase I human endotoxin trial evaluating recombinant human activated protein C (drotrecogin alfa [activated]). Data Extraction and Synthesis:The protein C pathway is positioned at the interface between the endothelium and the leukocyte response of the innate immune system. Activated protein C (APC) possesses profibrinolytic, anti-inflammatory, and anti-apoptotic properties, acting as an endothelial cell and microvascular modulator in opposition to thrombin and the proinflammatory cytokines. Distribution of the receptor for APC, endothelial protein C receptor, was detected on effector cells of the innate immune response. This suggests a further role for the protein C pathway in regulating inflammation. In neutrophils and eosinophils, an endothelial protein C receptor–mediated APC response leads to reduced migration in response to cytokine gradients. Endothelial protein C receptor may also suppress the apoptotic response in monocytes and enhance the expression of the adhesion integrin CD11b in granulocytes. The microvascular, anti-inflammatory influence of APC in sepsis is supported by suppression of endothelial cell adhesion molecules and the ability of APC to protect the endothelium from inflammatory insult. Conclusions:The coordinated effects of the protein C pathway on the endothelium and the leukocyte response of the innate immune system are supported by potential restriction of endothelial protein C receptor expression to cells of the innate immune system and by suppression of adhesion molecule expression on the endothelium by APC. Reduced neutrophil migration in response to cytokines is also mediated by endothelial protein C receptor. Further clinical studies will be needed to define the intrinsic role of the protein C pathway in coordinating the innate immune response in endothelium-based inflammation.

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.

Evaluation of protein C and other biomarkers as predictors of mortality in a rat cecal ligation and puncture model of sepsis.

Objective:To evaluate protein C and other factors associated with the septic response as predictors of mortality in a clinically relevant animal model of sepsis. Design:Laboratory investigation. Setting:Eli Lilly and Company discovery research laboratory. Subjects:Forty female Sprague Dawley Rats weighing 245–265 g. Interventions:Polyethylene catheters were surgically implanted into the femoral vein and sepsis was induced by cecal ligation and puncture (CLP). A solution of 5% dextrose in 0.9% saline was continuously infused via femoral catheters immediately following surgery. Blood sampling was done before surgery and at 6 and 20 hrs after surgery. Rats were then monitored for survival out to 4 days. Measurements and Main Results:Blood collections were used to measure blood glucose, bacteremia, plasma protein C, D-dimer, hormones, chemokines, cytokines, and myoglobin (as a marker of organ damage). Mortality was categorized into three groups: early death (before 30 hrs post-CLP), late death (after 30 hrs post-CLP), and survivors (96 hrs post-CLP). Compared with survivors, early death rats had statistically significant differences in 30 variables indicative of severe inflammation, coagulopathy, and muscle damage including less bacterial clearance, hypoglycemia, lower plasma protein C, higher plasma D dimer, higher plasma cytokine/ chemokines, and higher plasma myoglobin concentrations. Twenty variables had a moderate to strong correlation with time of death. Receiver operator characteristic curves generated from a simple logistic regression model indicated that KC and macrophage inflammatory protein-2, rodent homologues of the human growth related oncogene CXC chemokine family, and protein C were the best predictors of mortality in this model. Conclusions:The data from this study indicate that an early decrease in protein C concentration predicts poor outcome in a rat sepsis model. The data further indicate that increases in the CXC chemokines macrophage inflammatory protein-2 and KC precede poor outcome.

Activated Protein C Decreases Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand by an EPCR- Independent Mechanism Involving Egr-1/Erk-1/2 Activation

Background—APC is an antithrombotic and antiinflammatory serine protease that plays an important role in vascular function. We report that APC can suppress the proapoptotic mediator TRAIL in human umbilical vein endothelial cells, and we have investigated the signaling mechanism. Methods and Results—APC inhibited endothelial TRAIL expression and secretion and its induction by cell activation. To explore the mechanism, we examined factors associated with TRAIL regulation and demonstrated that APC increased the level of EGR-1, a transcriptional factor known to suppress the TRAIL promoter. APC also induced a significant increase in phosphorylation of ERK-1/2, required to activate EGR-1 expression. Activation of ERK-1/2 was dependent on the protease activated receptor-1 (PAR-1), but independent of the endothelial protein C receptor (EPCR). Using siRNA, we found that the effect of APC on the EGR-1/ERK signaling required for TRAIL inhibition was dependent on the S1P1 receptor and S1P1 kinase. Conclusions—Our data suggest that APC may provide cytoprotective activity by activating the ERK pathway, which upregulates EGR-1 thereby suppressing the expression of TRAIL. Moreover, we provide evidence that APC can induce a cell signaling response through a PAR-1/S1P1-dependent but EPCR-independent mechanism.

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.