Raul A. DeLa Cadena

Selective plasma kallikrein inhibitor attenuates acute intestinal inflammation in lewis rat

A specific plasma kallikrein inhibitor, Bz-Pro-Phe-boroArg (P8720), was used to define the relationship between the kallikrein-kinin (K-K) system and acute intestinal inflammation induced by bacterial peptidoglycan-polysaccharide (PG-APS) in Lewis rats. Group I received human serum albumin (HSA) intramurally in the intestine and was treated with HSA. Group II received PG-APS and was treated with P8720. Group III received PG-APS and was treated with HSA. P8720 attenuated the decrease of high-molecular-weight kininogen and factor XI activity (group II vs group III,P<0.01). P8720 therapy significantly but modestly decreased acute intestinal inflammation measured by gross gut score (P<0.01) and more dramatically reduced the tissue myeloperoxidase activity (P<0.05), a measure of granulocyte recruitment, in group II compared with group III. We conclude that the K-K system is directly involved in the pathogenesis of the acute phase of experimental acute inflammation. A specific inhibitor may modulate inflammatory bowel disease.

Clopidogrel, a P2Y12 receptor antagonist, potentiates the inflammatory response in a rat model of peptidoglycan polysaccharide-induced arthritis.

The P2Y12 receptor plays a crucial role in the regulation of platelet activation by several agonists, which is irreversibly antagonized by the active metabolite of clopidogrel, a widely used anti-thrombotic drug. In this study, we investigated whether reduction of platelet reactivity leads to reduced inflammatory responses using a rat model of erosive arthritis. We evaluated the effect of clopidogrel on inflammation in Lewis rats in a peptidoglycan polysaccharide (PG-PS)-induced arthritis model with four groups of rats: 1) untreated, 2) clopidogrel-treated, 3) PG-PS-induced, and 4) PG-PS-induced and clopidogrel-treated. There were significant differences between the PG-PS+clopidogrel group when compared to the PG-PS group including: increased joint diameter and clinical manifestations of inflammation, elevated plasma levels of pro-inflammatory cytokines (IL-1 beta, interferon (IFN) gamma, and IL-6), an elevated neutrophil blood count and an increased circulating platelet count. Plasma levels of IL-10 were significantly lower in the PG-PS+clopidogrel group compared to the PG-PS group. Plasma levels of platelet factor 4 (PF4) were elevated in both the PG-PS and the PG-PS+clopidogrel groups, however PF4 levels showed no difference upon clopidogrel treatment, suggesting that the pro- inflammatory effect of clopidogrel may be due to its action on cells other than platelets. Histology indicated an increase in leukocyte infiltration at the inflammatory area of the joint, increased pannus formation, blood vessel proliferation, subsynovial fibrosis and cartilage erosion upon treatment with clopidogrel in PG-PS-induced arthritis animals. In summary, animals treated with clopidogrel showed a pro-inflammatory effect in the PG-PS-induced arthritis animal model, which might not be mediated by platelets. Elucidation of the mechanism of clopidogrel-induced cell responses is important to understand the role of the P2Y12 receptor in inflammation.

Structure and functions of human kininogens

Evidence has accumulated over the past three decades implicating plasma kininogens in numerous inflammatory processes. Delineation of the detailed biochemistry and, more recently, the molecular biology of the human kininogens has resulted in a deeper understanding of the structure-function correlations of the human kininogens. Studies of alterations of human kininogens in disease states have yielded information about the mechanisms of their involvement in inflammatory states. Here, Raul DeLa Cadena and Robert Colman summarize kininogen function in relation to structure and diagnostic and therapeutic potential.

Coagulation Systems and Neutrophils in the Activation of Plasma Contact and Active Phase of Ulcerative Colitis

We have shown that the contact (kallikrein-kinin) system is involved in the pathogenesis ofexperimental enterocolitis. We now investigateactivation of the contact and coagulation pathways,platelets, and neutrophils in active and inactiveulcerative colitis patients as compared to normalcontrols. In active ulcerative colitis patients, asignificant decrease of plasma prekallikrein, highmolecular weight kininogen, and C1 inhibitor levels was observedas compared with controls, as well as prekallikreinactivation on western blots. Significant elevation ofprothrombin fragment (F1 + 2), which indicates thrombin generation, and elastase-α1-antitrypsincomplexes, reflecting neutrophil activation, were foundin patients with active disease. Plasmabeta-thromboglobulin, a marker of platelet activation,was elevated in both active and inactive disease and appearsto be a feature of ulcerative colitis. Activation ofcontact and coagulation pathways, as well asneutrophils, may mediate inflammation in the activephase of ulcerative colitis.We have shown that the contact (kallikrein-kinin) system is involved in the pathogenesis ofexperimental enterocolitis. We now investigateactivation of the contact and coagulation pathways,platelets, and neutrophils in active and inactiveulcerative colitis patients as compared to normalcontrols. In active ulcerative colitis patients, asignificant decrease of plasma prekallikrein, highmolecular weight kininogen, and C1 inhibitor levels was observedas compared with controls, as well as prekallikreinactivation on western blots. Significant elevation ofprothrombin fragment (F1 + 2), which indicates thrombin generation, and elastase-α1-antitrypsincomplexes, reflecting neutrophil activation, were foundin patients with active disease. Plasmabeta-thromboglobulin, a marker of platelet activation,was elevated in both active and inactive disease and appearsto be a feature of ulcerative colitis. Activation ofcontact and coagulation pathways, as well asneutrophils, may mediate inflammation in the activephase of ulcerative colitis.

The plasma kallikrein-kinin system in sepsis, inflammatory arthritis, and enterocolitis

The action of kinins mimics many of the characteristics of the inflammatory state, including increased capillary permeability, edema, pain, and vasodilation. It is logical that kinins play an important role in the systemic inflammatory response syndrome (1), which includes sepsis and trauma, as well as in localized processes, such as rheumatoid arthritis and inflammatory bowel disease. This article will consider the clinical and experimental evidence for the participation of the plasma Inflammation is a complex process involving the participation and interaction of plasma protease cascades, such as complement, coagulation, and fibrinolysis, and cells in the intravascular compartment, such as phagocytes (neutrophils and monocytes), hemostatic cells (platelets and endothelial cells), and immune cells (T and B lymphocytes). Moreover, each of these cells secretes inflammatory molecules, such as proteases (elastase and cathepsins), prostaglandins (prostacyclin and thromboxane A2), leukotrienes, platelet-activating factor, and a growing list of cytokines. It is thus unreasonable to believe that any one system can play a predominant role in inflammation. Nonetheless, both clinical and controlled experimental studies have indi-

Amelioration of inflammation, angiogenesis and CTGF expression in an arthritis model by a TSP1-derived peptide treatment.

Objective: To evaluate the effect of a thrombospondin 1 (TSP1)‐derived peptide on inflammation and angiogenesis in an animal model of erosive arthritis and to assess the relationship between TSP1 and connective tissue growth factor (CTGF) in the pathophysiology of rheumatoid arthritis. Methods: Erosive arthritis in Lewis rats was induced by peptidoglycan‐polysaccharide (PG‐PS). Animals were divided into four groups: (1) negative control and groups receiving, (2) no treatment, (3) treatment with a TSP1‐derived peptide, and (4) treatment with a scrambled peptide. Samples obtained from ankle joint, spleen and liver were studied using histology, histomorphometry, immunohistochemistry and RT‐PCR. Results: Histological data indicated that the TSP1‐derived peptide treatment decreased neovascularization, leukocyte infiltration and thickening of the synovial lining of the joint, and reduced granuloma formation in the spleen and liver when compared to control groups. Higher concentrations of CTGF and TSP1 proteins were observed in the affected areas of animals which did not receive TSP1‐derived peptide treatment. Also, immunofluorescence and RT‐PCR analyses showed an increase in CTGF protein expression and regulation, respectively, in the tissues of untreated animals when compared to the TSP1‐derived peptide treated animals. By immunofluorescence, TSP1 expression was decreased in the TSP1‐derived peptide treated animals. Moreover, macrophage/monocyte‐specific staining revealed a decrease in cell infiltration in the articular tissue of the TSP1‐derived peptide treated animals. Conclusion: Both inflammation and angiogenesis were decreased after TSP1‐derived peptide treatment indicating a potential pathway by which TSP1 interaction with neutrophils induces CTGF in RA affected tissues. J. Cell. Physiol. 211: 504–512, 2007.

Thrombospondin-1 and transforming growth factor beta are pro-inflammatory molecules in rheumatoid arthritis

Thrombospondin-1 (TSP1/THBS1) plays a major role in the pathophysiology of rheumatoid arthritis (RA); however, its interface with the cytokine network involved in RA has not been delineated. Correlations were performed between plasma levels of TSP1 and selected cytokines from blood samples collected from 20 patients affected by RA and 13 healthy donors (control). Plasma levels of TSP1 and tissue growth factor beta (TGFbeta) were determined by standard enzyme-linked immunosorbent assay, and cytokines were measured by protein profiling rolling-circle amplification (RCA). TSP1 circulating levels in plasma were found significantly increased in the RA patients when compared with control individuals (P = 0.039). The plasma levels of TGFbeta were also increased in the RA patients, which indicates a statistical trend. Cytokine levels of interleukin (IL)-4, IL-5, IL-12, chemokine CXC 10 (CXCL10/IP10), and chemokine CC 4 (CCL4)/MIP1beta were significantly increased in the RA patients when compared with the control group. In summary, this study demonstrates increased plasma levels of TSP1, which correlated with increased levels of proinflammatory cytokines in plasma of RA patients. More detailed research is required to explore the cytokine imprint yielded by this study and its interface with TSP1 and TGFbeta.

The Axis of Thrombospondin-1, Transforming Growth Factor Beta and Connective Tissue Growth Factor: An Emerging Therapeutic Target in Rheumatoid Arthritis

Biologic therapy for rheumatoid arthritis (RA) targets specific molecules that mediate and sustain the clinical manifestations of this complex illness. Compared with the general population, patients with RA die prematurely, in part due to associated cardiovascular disease. Even though the mechanisms by which premature atherosclerosis develops in RA is unknown, chronic inflammation may play a major role. This review connects current knowledge of the pathophysiology of RA with data available in the literature related to thrombospondin-1 (TSP1), transforming growth factor beta (TGFbeta and connective tissue growth factor (CTGF) and their relationship with cardiovascular disease in RA. The TSP1/TGFbeta/CTGF axis may contribute in the pro-inflammatory and pro-atherogenic state in patients affected with RA. In fact, increased TSP1 plasma levels are found in patients of RA. TGFbeta is activated by TSP1 through a non-enzymatic mechanism and is constitutively overexpressed by synovial fibroblasts from RA patients. Activation of TGFbeta pathway in synovial fibroblasts and other cells including neutrophils leads to downstream upregulation of CTGF. Overexpression of CTGF is associated with angiogenesis, fibrosis, atherosclerotic blood vessels and erosive arthritis lesions. Recent RA therapies emphasize the need for aggressive control of the activity of the disease to prevent premature atherosclerosis in RA patients. The complexity and heterogeneity of RA as judged by response to a wide spectrum of treatments mandates the elucidation of unknown pro-inflammatory pathways playing a major role in this disease. The TSP1/TGFbeta/CTFG axis represents one of these pro-inflammatory pathways that may result in the development of promising therapeutic strategies to prevent chronic inflammation and thus premature atherosclerosis in RA.

A Monoclonal Antibody to High-Molecular Weight Kininogen Is Therapeutic in a Rodent Model of Reactive Arthritis

We reported that high-molecular weight kininogen is proangiogenic by releasing bradykinin and that a monoclonal antibody to high-molecular weight kininogen, C11C1, blocked its binding to endothelial cells. We now test if this antibody can prevent arthritis and systemic inflammation in a Lewis rat model. We studied 32 animals for 16 days. Group I (negative control) received saline intraperitoneally. Group II (disease-treated) received peptidoglycan-polysaccharide simultaneously with C11C1. Group III (disease-untreated) received peptidoglycan-polysaccharide simultaneously with isotype-matched mouse IgG. Group IV (disease-free-treated) and group V (disease-free isotype-treated) received saline and C11C1 or mouse IgG. Analysis of joint diameter changes showed a decrease in the C11C1 disease-treated group compared to the disease-untreated group. The hind paw inflammatory score showed a decrease in the intensity and extent of inflammation between the disease-untreated and the C11C1 disease-treated group. Prekallikrein, high-molecular weight kininogen, factor XI, and factor XII were decreased in the disease-untreated group compared to the C11C1 disease-treated group. T-kininogen was increased in the disease-untreated group when compared with the C11C1 disease-treated group. Disease-free groups IV and V did not show any sign of inflammation at any time. This study shows that monoclonal antibody C11C1 attenuates plasma kallikrein-kinin system activation, local and systemic inflammation, indicating therapeutic potential in reactive arthritis.

Three noncontiguous peptides comprise binding sites on high-molecular-weight kininogen to neutrophils

The binding of high-molecular-weight kininogen (HK) to neutrophils (polymorphonuclear leukocytes, PMN) is required for the stimulation of aggregation and degranulation by human plasma kallikrein as well as the displacement of fibrinogen from this cell surface. The putative receptor for HK is the leukocyte integrin alphaMbeta2, and domains 3 (D3) and 5 (D5) of HK form its binding site. To further map the binding sites on HK for PMN, we used D3 recombinant exon products and designed peptides from D3 and D5. In D3, a heptapeptide, Leu271-Ala277, from exon 7 product, and a peptide, Cys333-Cys352, from exon 9 product can inhibit binding of kininogen to PMN. Two contiguous peptides from D5 in the histidine-glycine-rich region, Gly442-Lys458 and Phe459-Lys478, each inhibit the binding of HK to PMN. This study has thus delineated three noncontiguous surface-oriented sequences on HK, which together comprise all or most of the binding site for human PMN.The binding of high-molecular-weight kininogen (HK) to neutrophils (polymorphonuclear leukocytes, PMN) is required for the stimulation of aggregation and degranulation by human plasma kallikrein as well as the displacement of fibrinogen from this cell surface. The putative receptor for HK is the leukocyte integrin αMβ2, and domains 3 (D3) and 5 (D5) of HK form its binding site. To further map the binding sites on HK for PMN, we used D3 recombinant exon products and designed peptides from D3 and D5. In D3, a heptapeptide, Leu271-Ala277, from exon 7 product, and a peptide, Cys333-Cys352, from exon 9 product can inhibit binding of kininogen to PMN. Two contiguous peptides from D5 in the histidine-glycine-rich region, Gly442-Lys458and Phe459-Lys478, each inhibit the binding of HK to PMN. This study has thus delineated three noncontiguous surface-oriented sequences on HK, which together comprise all or most of the binding site for human PMN.