Biomedical subjects

Lawrence A Potempa

Publications and source records attributed to Lawrence A Potempa.

12 recordsLinked to original sources

PubMed2006

Cell membranes and liposomes dissociate C-reactive protein (CRP) to form a new, biologically active structural intermediate: mCRP(m).

Emerging evidence indicates that C-reactive protein (CRP) has at least two conformationally distinct isoforms, i.e., pentameric CRP (pCRP) and monomeric CRP (mCRP or CRP subunit). Both CRP isoforms are proposed to play roles in inflammation and may participate in the pathogenesis of cardiovascular disease. However, the origin of mCRP in situ and the interplay between the two CRP isoforms under physiological/pathological circumstances remain elusive. Herein, by probing conformational alteration, neoepitope expression, and direct visualization using electron-microscopy, we have shown that calcium-dependent binding of pCRP to membranes, including liposomes and cell membranes, led to a rapid but partial structural change, producing molecules that express CRP subunit antigenicity but with retained native pentameric conformation. This hybrid molecule is herein termed mCRP(m). The formation of mCRP(m) was associated with significantly enhanced complement fixation. mCRP(m) can further detach from membrane to form the well-recognized mCRP isoform converted in solution (mCRP(s)) and exert potent stimulatory effects on endothelial cells. The membrane-induced pCRP dissociation not only provides a physiologically relevant scenario for mCRP formation but may represent an important mechanism for regulating CRP function.

Animals↗

PubMed2006

Effect of modified C-reactive protein on complement activation: a possible complement regulatory role of modified or monomeric C-reactive protein in atherosclerotic lesions.

OBJECTIVE: The capacity of human C-reactive protein (CRP) to activate/regulate complement may be an important characteristic that links CRP and inflammation with atherosclerosis. Recent advances suggest that in addition to classical pentameric CRP, a conformationally distinct isoform of CRP, termed modified or monomeric CRP (mCRP), may also play an active role in atherosclerosis. Although the complement activation behavior of CRP has been well established, the capacity of mCRP to interact with and activate the complement cascade is unknown. METHODS AND RESULTS: mCRP bound avidly to purified C1q, and this binding occurred primarily through collagen-like region of C1q. Fluid phase mCRP inhibited the activation of complement cascade via engaging C1q from binding with other complement activators. In contrast, when immobilized or bound to oxidized or enzymatically modified low-density lipoprotein, mCRP could activate classical complement pathway. Low-level generation of sC5b-9 indicated that the activation largely bypassed the terminal sequence of complement, which appears to involve recruitment of Factor H. CONCLUSIONS: These results indicate that mCRP can both inhibit and activate the classical complement pathway by binding C1q, depending on whether it is in fluid phase or surface-bound state.

Atherosclerosis↗

PubMed2006

C-reactive protein: a family of proteins to regulate cardiovascular function.

Based on a multitude of clinical studies, C-reactive protein (CRP) has emerged as a risk marker for the development of cardiovascular disease, leading to recently published recommendations for screening the general population for plasma CRP level as a predictor for future cardiovascular events. However, uncertainties exist in how to apply these recommendations to populations with very high serum CRP levels and a high prevalence of cardiovascular disease, such as patients with end-stage renal disease. Furthermore, in vitro results are conflicting concerning the role of CRP in the vessel wall. Although many data are in favor of a proinflammatory effect of CRP, evidence is accumulating that CRP also exerts anti-inflammatory actions, mainly in neutrophils and platelets. Many of the apparently contradictory actions of CRP may be attributed to method issues, but, of interest, also may be explained by the existence of 2 distinct conformations of CRP, the native pentamer (nCRP) and modified CRP (mCRP) forms. nCRP is the classical acute-phase reactant detected in serum, whereas mCRP represents a predominantly tissue-bound form. It is detected immunohistochemically, mainly in and around endothelial and vascular smooth muscle cells. Although mCRP activates endothelial cells and neutrophils, induces neutrophil adhesion to the endothelium, and delays apoptosis of neutrophils in vitro, these effects were absent using nCRP. Clearly defined CRP conformers thus may provide a tool for how to reconcile the reported proinflammatory and anti-inflammatory properties of CRP. There is good evidence to believe that CRP is more than just a "bad guy," and further experiments are needed to determine how these 2 configurations contribute to atherogenesis, development of cardiovascular disease, and acute coronary events.

Animals↗

PubMed2005

Interactions of C-reactive protein with low-density lipoproteins: implications for an active role of modified C-reactive protein in atherosclerosis.

The interaction of C-reactive protein with low-density lipoprotein is considered to be one of the key properties that link C-reactive protein with atherosclerosis. However the data obtained to date are controversial, and hence make it difficult to conclude actual physiological or pathological impact of such interaction. The incompatible findings could be ascribed to the different structural state of C-reactive protein and/or low-density lipoprotein. We investigated in detail the interaction of various C-reactive protein isoforms with native and modified low-density lipoprotein. Our data showed "C-reactive protein" could indeed interact with each of native low-density lipoprotein, oxidized or enzymatically modified low-density lipoprotein, but that interaction occurs primarily when C-reactive protein is conformed in a modified form and not pentameric structure. Low level of modified C-reactive protein "contaminant" could confer C-reactive protein obvious low-density lipoprotein binding capacity. Interaction of modified C-reactive protein and low-density lipoprotein was mediated synergistically by both electrostatic association with ApoB and hydrophobic insertion into lipid layer. When complexed with modified C-reactive protein, macrophage binding/uptake of native and oxidized low-density lipoprotein was either increased 150% or decreased 35%, respectively. Thus the interaction of modified C-reactive protein with low-density lipoprotein may contribute to the regulation of low-density lipoprotein metabolism and foam cell formation in arterial wall. These results highlight an active role of modified C-reactive protein in atherosclerotic process.

Atherosclerosis↗

PubMed2005

Loss of pentameric symmetry in C-reactive protein induces interleukin-8 secretion through peroxynitrite signaling in human neutrophils.

Plasma levels of C-reactive protein (CRP), nitrotyrosine, and interleukin-8 (IL-8) are known predictors of acute cardiovascular events. Peroxynitrite (ONOO-) may function as an intracellular signal for the production of IL-8; however, it is not known whether CRP regulates these events. Emerging evidence suggests that some bioactivities of CRP are expressed only when the pentameric structure of CRP is lost, resulting in formation of monomeric or modified CRP (mCRP). We studied the impact of human native CRP and bioengineered mCRP that cannot rearrange into the pentameric structure on ONOO- formation and ONOO--mediated IL-8 gene expression in human leukocytes. Incubation of human whole blood or isolated neutrophils with mCRP (0.1 to 100 microg/mL) for 4 hours increased IL-8 gene expression and secretion that was blocked approximately 70% by the NO synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME). In neutrophils, mCRP simultaneously increased superoxide production and endothelial nitric oxide synthase-mediated NO formation, leading to enhanced ONOO- formation, and consequently activation of nuclear factor-kappaB and activator protein-1. Native CRP had no detectable effect at 4 hours, whereas it enhanced IL-8 release after a 24-hour incubation that was blocked by L-NAME. An anti-CD16 antibody, but not an anti-CD32 antibody, produced 60% to 70% reductions in mCRP-stimulated NO formation and IL-8 release (both P<0.05). These results suggest that loss of the pentameric symmetry in CRP, resulting in formation of mCRP, leads to IL-8 release from human neutrophils via peroxynitrite-mediated activation of nuclear factor-kappaB and activator protein-1.

C-Reactive Protein↗

PubMed2005

Native C-reactive protein increases whereas modified C-reactive protein reduces atherosclerosis in apolipoprotein E-knockout mice.

BACKGROUND: C-reactive protein (CRP) may have proatherogenic but also vasoprotective properties. We tested the hypothesis that the configuration of CRP (pentameric, or native [nCRP], versus monomeric, or modified [mCRP]) determines these different characteristics in an in vivo model. METHODS AND RESULTS: We investigated the effects of human nCRP and mCRP on the development of atherosclerosis in apolipoprotein E-knockout (ApoE(-/-)) mice. Treatment with nCRP for 8 weeks (2.5 mg/kg SC weekly) resulted in a 4-fold-higher mean aortic plaque area in 14-week-old female ApoE(-/-) mice compared with the saline controls. In contrast, mean plaque size was decreased by approximately 50% in mCRP-treated ApoE(-/-) mice (2.5 mg/kg SC weekly). Using immunohistochemistry, we report the natural presence of the mCRP antigen in saline controls. mCRP antigen was expressed in smooth muscle cells and extracellularly in the vicinity of the plaques to a similar level in both CRP-treated groups and saline controls. mCRP and ApoB colocalized with macrophages and were equally upregulated in all aortic plaques. Vascular cell adhesion molecule expression was increased, and CD154 and intercellular adhesion molecule showed a trend for higher expression in nCRP-treated compared with mCRP-treated mice. CD154 expression in the vessel wall and plaque size correlated significantly. mCRP-treated ApoE(-/-) exhibited higher serum levels of the antiinflammatory interleukin-10 compared with the other 2 groups. CONCLUSIONS: Here, we show that mCRP and nCRP have opposite effects on atherosclerosis in ApoE(-/-) mice. These data may explain in part the conflicting activities previously reported for CRP in models of atherogenesis.

Animals↗

PubMed2005

Native and modified C-reactive protein bind different receptors on human neutrophils.

Native C-reactive protein (CRP) is a planar pentamer of identical subunits expressed at high serum levels during the acute phase of inflammation. At inflammatory sites, an isomeric form termed modified CRP (mCRP) is expressed and reveals neoantigenic epitopes associated with modified monomeric CRP subunits. mCRP cannot assume the native pentameric conformation but rather forms a nonpentameric aggregate of monomers. While native CRP inhibits neutrophil movement in vitro and in vivo, the effect of mCRP on neutrophil movement has not been reported. To model the biological function and biochemical interaction of mCRP on neutrophils, in vitro chemotaxis and binding experiments were performed using mCRP. Reported here, mCRP effectively inhibited fMLP-induced chemotaxis similar to native CRP. Additionally, mCRP increased binding of labeled native CRP to neutrophils. This increased binding occurred by direct protein-protein interaction of the C-terminus thereby implicating the CRP(199-206) sequence. Binding of mCRP to neutrophils was blocked by anti-CD16 monoclonal antibody whereas native CRP was not. These results suggest that modified forms of CRP inhibit chemotaxis, a function similar to native CRP, but that mCRP and native molecule bind different receptors on human neutrophils.

Animals↗

PubMed2005

Production of modified C-reactive protein in U937-derived macrophages.

Plasma C-reactive protein (CRP) has been proposed to be a strong independent predictor for cardiovascular disease. This circulating form of CRP (native CRP or nCRP) is well described. Recently, the existence of a conformationally distinct isoform of CRP (modified CRP or mCRP) has been reported. The relevance of each CRP isoform to atherosclerotic disease is unknown. The purpose of this study was to examine the natural expression of CRP in undifferentiated, differentiated, and stimulated macrophages, cells known to contribute to atherogenesis in vivo, and to determine whether transcribed CRP was expressed as nCRP or mCRP. Macrophages were generated from U937 monocytes using phorbol 12-myristate 13-acetate. Differentiated macrophages were further stimulated with lipopolysaccharides (LPS). In undifferentiated, differentiated, and stimulated cells, CRP expression was assessed by reverse transcription-polymerase chain reaction, and CRP protein production was measured by fluorescence microscopy and flow cytometry (cellular CRP) or high-sensitivity enzyme-linked immunosorbent assay (secreted CRP). CRP transcript was minimally expressed in undifferentiated cells. Expression increased markedly in macrophages during differentiation and was not affected by LPS at 24 hrs. Cellular CRP protein increased in a time-dependent manner after LPS stimulation, and this induction was mediated via interleukin (IL)-6 and IL-1beta. A small amount of secreted CRP was detected in the media of differentiated cells, but it was not significantly increased after LPS stimulation. Using specific monoclonal antibodies, our data indicate that cellular CRP is directly translated as the mCRP rather than the nCRP isomer. These results indicate that U937-derived macrophages are a good cell model to further study the production of mCRP under conditions relevant for the atherogenic process.

Acute-Phase Reaction↗

PubMed2004

Opposing effects of C-reactive protein isoforms on shear-induced neutrophil-platelet adhesion and neutrophil aggregation in whole blood.

BACKGROUND: Plasma C-reactive protein (CRP) level is a powerful predictor of cardiovascular events. However, it is not known whether CRP could affect neutrophil-platelet adhesion and neutrophil aggregation, key events in acute coronary syndromes. Emerging in vitro evidence suggests that some bioactivities of CRP are expressed on loss of the pentameric symmetry, resulting in formation of modified or monomeric CRP (mCRP). METHODS AND RESULTS: We studied the impact of human native CRP and bioengineered mCRP that cannot rearrange into the pentameric structure on the kinetics of neutrophil-platelet adhesion and neutrophil aggregation in whole blood subjected to shear (approximately 100 s(-1)) using real-time flow cytometry. Shear resulted in upregulation of platelet P-selectin expression, leading to platelet capture of neutrophils and subsequent neutrophil aggregation, which was dependent on P-selectin, L-selectin, and CD18. Native CRP at clinically relevant concentrations markedly attenuated these changes. The residual amount of neutrophil adhesion was blocked with anti-CD18 or anti-CD11b antibody. By contrast, mCRP concentration-dependently enhanced shear-induced platelet P-selectin expression and increased the rate and extent of formation of both neutrophil-platelet and neutrophil-neutrophil aggregates. Complete abrogation of platelet-neutrophil adhesion and neutrophil aggregation required both anti-P-selectin and anti-CD18 antibodies but not anti-L-selectin antibody. The CRP action was markedly inhibited by an anti-CD32 antibody, whereas the mCRP effects were significantly attenuated by an anti-CD16 antibody. CONCLUSIONS: These results indicate that native CRP inhibits platelet activation and prevents platelet capture of neutrophils, whereas mCRP displays potent prothrombotic activities under low levels of shear. Thus, mCRP rather than native CRP may precipitate acute coronary syndromes.

Antigens, CD↗

PubMed2004

Conformational rearrangement in C-reactive protein is required for proinflammatory actions on human endothelial cells.

BACKGROUND: C-reactive protein (CRP) has been suggested to actively amplify the inflammatory response underlying coronary heart diseases by directly activating endothelial cells. In this study, we investigated whether loss of the cyclic pentameric structure of CRP, resulting in formation of modified or monomeric CRP (mCRP), is a prerequisite for endothelial cell activation. METHODS AND RESULTS: We examined the impact of native CRP and mCRP on the production of monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8), key regulators of leukocyte recruitment, and on the expression of intercellular adhesion molecule-1 (ICAM-1), E-selectin, and vascular adhesion molecule-1 (VCAM-1) in human cultured coronary artery endothelial cells (HCAECs). Incubation with mCRP for 4 hours increased MCP-1 and IL-8 secretion and mRNA levels and expression of ICAM-1, E-selectin, and VCAM-1 protein and mRNA. Significant induction occurred at 1 to 5 microg/mL, reached a maximum at 30 microg/mL, and did not require the presence of serum. Native CRP was without detectable effects at 4 hours, whereas it enhanced cytokine release after a 24-hour incubation. An anti-FcgammaRIII (CD16) but not an anti-FcgammaRII (CD32) antibody produced a 14% to 32% reduction of the mCRP effects (P<0.05). mCRP but not CRP evoked phosphorylation of p38 mitogen-activated protein kinase, and inhibition of this kinase with SB 203580 reversed the effects of mCRP. Furthermore, culture of HCAECs in the presence of SB203580 markedly decreased mCRP-stimulated E-selectin and ICAM-1-dependent adhesion of neutrophils to HCAECs (P<0.001). CONCLUSIONS: Loss of pentameric symmetry in CRP, resulting in formation of mCRP, promotes a proinflammatory HCAEC phenotype through a p38 MAPK-dependent mechanism.

C-Reactive Protein↗

PubMed2003

Tubular staining of modified C-reactive protein in diabetic chronic kidney disease.

BACKGROUND: Serum levels of C-reactive protein (CRP) increase during various atherosclerotic as well as kidney diseases. Whether CRP plays a pathophysiological role or rather serves as a marker is unknown. Here, we investigated the role of CRP in diabetic patients with chronic kidney disease. METHODS: Kidney biopsies from 20 diabetic patients, six with IgA nephropathy and six controls (absence of disease) were stained using a commercially available anti-CRP antibody (clone 8). We characterized this antibody by ELISA and found that it mainly recognized 'modified' CRP (mCRP), the conformational isoform of CRP that occurs after dissociation of the pentameric isomer. RESULTS: A specific CRP signal was observed in the cytoplasma of tubules in 17 out of 20 kidney biopsies from diabetic patients, while all glomeruli, vessels and interstitium stained CRP-negative. This signal was absorbed against the mCRP protein suggesting that the detected tissue-based antigen is more closely related to the mCRP conformer than to the native CRP conformer. Almost all patients (eight out of nine) with severe chronic kidney disease [glomerular filtration rate (GFR) <30 ml/min/1.73 m(2)] strongly stained for the mCRP antigen, whereas only four out of 11 patients with mild and moderate chronic kidney disease (GFR >/=30 ml/min/1.73 m(2)) demonstrated a strong CRP signal. Normal renal tissue and most biopsies with IgA nephropathy were mCRP negative. Severity of histologic changes as assessed by histology score and mCRP staining correlated significantly, but no correlation was evident between tubular mCRP staining and serum levels of CRP or proteinuria. CONCLUSIONS: The present group of diabetic patients showed progressive tubular mCRP staining with declining renal function and increasing severity of histological lesions. Further studies in less proteinuric patients should clarify whether tubular mCRP expression constitutes a progression factor. It also needs to be demonstrated whether mCRP accumulates in tubuli to further stimulate interstitial fibrosis or is mandatory for the resolution of the process. Since mCRP staining was independent of proteinuria we suggest that mCRP is locally produced.

Adolescent↗

PubMed2002

Loss of pentameric symmetry of C-reactive protein is associated with delayed apoptosis of human neutrophils.

Human neutrophil granulocytes die rapidly, and their survival is contingent upon rescue from programmed cell death by signals from the environment. Here we report that a novel signal for delaying neutrophil apoptosis is the classic acute phase reactant, C-reactive protein (CRP). However, this anti-apoptotic activity is expressed only when the cyclic pentameric structure of CRP is lost, resulting in formation of modified or monomeric CRP (mCRP), which may be formed in inflamed tissues. By contrast, native pentameric CRP and CRP peptides 77-82, 174-185, and 201-206 failed to affect neutrophil apoptosis. The apoptosis delaying action of mCRP was markedly attenuated by an antibody against the low affinity IgG immune complex receptor (CD16) but not by an anti-CD32 antibody. mCRP evoked a transient concurrent activation of the extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase/Akt signaling pathways, leading to inhibition of caspase-3 and consequently to delaying apoptosis. Consistently, pharmacological inhibition of either ERK or Akt reversed the anti-apoptotic action of mCRP; however, they did not produce additive inhibition. Thus, mCRP, but not pentameric CRP or peptides derived from CRP, promotes neutrophil survival and may therefore contribute to amplification of the inflammatory response.

Adult↗