Biomedical subjects

A J Valente

Publications and source records attributed to A J Valente.

At least 19 recordsLinked to original sources

PubMed2001

Transcriptional regulation of the p67phox gene: role of AP-1 in concert with myeloid-specific transcription factors.

We have investigated the myeloid-specific transcriptional regulation of p67(phox), an essential component of phagocyte respiratory burst NADPH oxidase. Analysis was carried out on the p67(phox) 5'-flanking region from -3669 to -4 (relative to ATG), including the first exon and intron and part of the second exon. The construct extending from -985 to -4 produced the highest luciferase activity in myeloid HL-60 cells but was not active in HeLa or Jurkat cells, indicating myeloid-specific expression. Four active elements were identified: Sp1/Sp3 at -694, PU.1 at -289, AP-1 at -210, and PU.1/HAF1 at -182, the latter three being in the first intron. These cis elements bound their cognate transacting factors both in vitro and in vivo. Mutation of the Sp1, PU.1, or PU.1/HAF1 site each decreased promoter activity by 35-50%. Mutations in all three sites reduced promoter activity by 90%. However, mutation of the AP-1 site alone nearly abolished promoter activity. The AP-1 site bound Jun and Fos proteins from HL-60 cell nuclear extract. Co-expression with Jun B in AP-1-deficient cells increased promoter activity by 3-fold. These data show that full p67(phox) promoter activity requires cooperation between myeloid-specific and nonmyeloid transcription factors, with AP-1 being the most critical for function.

Base Sequence↗

PubMed2001

Agonist-specific regulation of monocyte chemoattractant protein-1 expression by cyclooxygenase metabolites in hepatic stellate cells.

Activated hepatic stellate cells (HSC) regulate the liver "wound-healing" response through expression of chemokines, including monocyte chemoattractant protein-1 (MCP-1), which participate in the formation of the inflammatory infiltrate during liver injury. Cyclooxygenase (COX) catalyzes the conversion of arachidonic acid into prostaglandins, which may contribute to the inflammatory response. In this study, we investigated the effects of COX inhibitors on the expression of MCP-1 in cultured HSC. Pretreatment of HSC with nonspecific COX inhibitors such as indomethacin or ibuprofen markedly reduced the expression of MCP-1 caused by exposure to tumor necrosis factor alpha (TNF-alpha) or interleukin-1alpha (IL-1alpha). NS-398, a specific COX-2 inhibitor, also resulted in a dose-dependent inhibition of MCP-1 gene and protein expression. These effects were dependent on reduced MCP-1 transcription, as established using a reporter plasmid. In contrast, the up-regulation of MCP-1 expression caused by interferon gamma (IFN-gamma) was not sensitive to COX inhibitors. Quiescent HSC did not show detectable expression of COX-2, which became evident after activation in culture, and while TNF-alpha and IL-1alpha markedly increased the expression of COX-2, IFN-gamma did not have any effects. Pretreatment of HSC with the stable cyclic adenosine monophosphate (cAMP) analog, 8-bromo cAMP, reverted the effects of the COX-2 inhibitor, but not of a nuclear factor-kappaB (NF-kappaB) inhibitor, demonstrating that prostaglandins modulate MCP-1 expression via production of cAMP. On the other hand, the action of NF-kappaB inhibitors was negligible in IFN-gamma-stimulated cells. These findings indicate that cross-talk between cytokines and a prostaglandin-cAMP pathway differentially regulates the proinflammatory potential of HSC, contributing to the modulation of liver tissue inflammation.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed2000

Evolution of human and non-human primate CC chemokine receptor 5 gene and mRNA. Potential roles for haplotype and mRNA diversity, differential haplotype-specific transcriptional activity, and altered transcription factor binding to polymorphic nucleotides in the pathogenesis of HIV-1 and simian immunodeficiency virus.

Polymorphisms in CC chemokine receptor 5 (CCR5), the major coreceptor of human immunodeficiency virus 1 (HIV-1) and simian immunodeficiency virus (SIV), have a major influence on HIV-1 transmission and disease progression. The effects of these polymorphisms may, in part, account for the differential pathogenesis of HIV-1 (immunosuppression) and SIV (natural resistance) in humans and non-human primates, respectively. Thus, understanding the genetic basis underlying species-specific responses to HIV-1 and SIV could reveal new anti-HIV-1 therapeutic strategies for humans. To this end, we compared CCR5 structure/evolution and regulation among humans, apes, Old World Monkeys, and New World Monkeys. The evolution of the CCR5 cis-regulatory region versus the open reading frame as well as among different domains of the open reading frame differed from one another. CCR5 cis-regulatory region sequence variation in humans was substantially higher than anticipated. Based on this variation, CCR5 haplotypes could be organized into seven evolutionarily distinct human haplogroups (HH) that we designated HHA, -B, -C, -D, -E, -F, and -G. HHA haplotypes were defined as ancestral to all other haplotypes by comparison to the CCR5 haplotypes of non-human primates. Different human and non-human primate CCR5 haplotypes were associated with differential transcriptional regulation, and various polymorphisms resulted in modified DNA-nuclear protein interactions, including altered binding of members of the NF-kappaB family of transcription factors. We identified novel CCR5 untranslated mRNA sequences that were conserved in human and non-human primates. In some primates, mutations at exon-intron boundaries caused loss of expression of selected CCR5 mRNA isoforms or production of novel mRNA isoforms. Collectively, these findings suggest that the response to HIV-1 and SIV infection in primates may have been driven, in part, by evolution of the elements controlling CCR5 transcription and translation.

Animals↗

PubMed1999

Critical flanking sequences of PU.1 binding sites in myeloid-specific promoters.

The myeloid-specific transcription factor PU.1 is essential for expression of p47(phox), a component of the superoxide-forming phagocyte NADPH oxidase. The consensus PU.1 binding sequence (GAGGAA) is located on the non-coding strand from position -40 to -45 relative to the transcriptional start site of the p47phox promoter. A promoter construct extending to -46 was sufficient to drive tissue-specific expression of the luciferase reporter gene, but extension of the promoter from -46 to -48 resulted in a significant increase in reporter expression. Mutations of the nucleotides G at -46 and/or T at -47 reduced both reporter expression and PU.1 binding, whereas mutations at -48 had no effect. The PU.1 binding avidity of these sequences correlated closely with their capacity to dictate reporter gene transcription. In parallel studies on the functional PU.1 site in the promoter of CD18, mutations of nucleotides G and T at positions -76 and -77 (corresponding to -46 and -47, respectively, of the p47phox promoter) reduced PU.1 binding and nearly abolished the contribution of this element to promoter activity. We conclude that the immediate flanking nucleotides of the PU.1 consensus motif have significant effects on PU.1 binding avidity and activity and that this region is the dominant cis element regulating p47phox expression.

Base Sequence↗

PubMed1999

The expression of monocyte chemoattractant protein-1 and other chemokines by osteoblasts.

Chemokines are low molecular weight secretory proteins that function principally as stimulators of leukocyte recruitment. There are four defined chemokine subfamilies based on their primary structure, CXC, CC, C and CX3C. Members of the CC chemokine subfamily, a such as monocyte chemoattractant protein 1 (MCP-1) are chemotactic for monocytes and other leukocyte subsets. Because monocytes produce factors that regulate bone formation or resorption, such as PDGF, IL-1 or TNF, chemokines that initiate their recruitment are likely to be important in regulating osseous metabolism. In the studies below, data is presented demonstrating mechanisms of MCP-1 expression in osteoblastic cells. These studies establish that MCP-1 is induced during osseous inflammation and in developmentally regulated bone remodelling, and is associated with enhanced monocyte recruitment when applied to osseous lesions.

Animals↗

PubMed1999

Regulation of low shear flow-induced HAEC VCAM-1 expression and monocyte adhesion.

We recently reported that prolonged exposure of human aortic endothelial cells (HAEC) to low shear stress flow patterns is associated with a sustained increase in the activated form of the transcriptional regulator nuclear factor-kappaB (NF-kappaB). Here we investigate the hypothesis that low shear-induced activation of NF-kappaB is responsible for enhanced expression of vascular cell adhesion molecule (VCAM-1) resulting in augmented endothelial cell-monocyte (EC-Mn) adhesion and that this activation is dependent on intracellular oxidant activity. Before exposure to low shear (2 dyn/cm2) for 6 h, HAEC were preincubated with or without the antioxidants pyrrolidine dithiocarbamate (PDTC) or N-acetyl-L-cysteine (NAC). PDTC strongly inhibited low shear-induced activation of NF-kappaB, expression of VCAM-1, and EC-Mn adhesion. Paradoxically, NAC exerted a positive effect on low shear-induced VCAM-1 expression and EC-Mn adhesion and only slightly downregulated NF-kappaB activation. However, cytokine-induced NF-kappaB activation and VCAM-1 expression are blocked by both PDTC and NAC. These data suggest that NF-kappaB plays a key role in low shear-induced VCAM-1 expression and that pathways mediating low shear- and cytokine-induced EC-Mn adhesion may be differentially regulated.

Acetylcysteine↗

PubMed1999

Regulated expression of MCP-1 by osteoblastic cells in vitro and in vivo.

Inflammation is characterized by the recruitment of leukocytes from the vasculature. Recent studies have implicated chemokines as an important class of mediators that function principally to stimulate leukocyte recruitment, and in some cases, leukocyte activity. There are four defined chemokine subfamilies based on their primary structure, CXC, CC, C and CX3C. Members of the CC chemokine subfamily, such as monocyte chemoattractant protein 1 (MCP-1), are chemotactic for monocytes and other leukocyte subsets. The studies described below focus on the expression of MCP-1 in vitro and in vivo in an osseous environment. These studies indicate that MCP-1 is typically not expressed in normal bone or by normal osteoblasts in vitro. Upon stimulation by inflammatory mediators, MCP-1 is up-regulated. This expression is temporally and spatially associated with the recruitment of monocytes in both osseous inflammation and during developmentally regulated bone remodelling. Furthermore, exogenous MCP-1 applied to inflamed bone enhances the recruitment of monocytes. Because monocytes produce factors that influence osseous metabolism, including but not limited to prostglandins, platelet-derived growth factor, interleukin-1 or tumor necrosis factor, chemokines that initiate their recruitment are likely to be highly important.

Animals↗

PubMed1998

A complex element regulates IFN-gamma-stimulated monocyte chemoattractant protein-1 gene transcription.

Monocyte chemoattractant protein-1 (MCP-1) is induced in chronic osseous inflammation, and is temporally and spatially correlated with monocyte recruitment. We investigated the mechanism of MCP-1 regulation in a human osteoblastic cell line in response to IFN-gamma, a potent mediator of the immune inflammatory response. Nuclear run-on and stability studies demonstrated that IFN-gamma stimulated MCP-1 transcription and did not enhance mRNA stabilization. Using MCP-1 promoter/reporter gene constructs, we determined that IFN-gamma-enhanced MCP-1 transcription is regulated by a 29-bp element located at -227 relative to the ATG start codon. This element contains a 13-bp CT-rich sequence (GCTTCCCTTTCCT) adjacent to a IFN-gamma activation site (GAS). Since deletion of the CT sequence enhanced both the magnitude and duration of IFN-gamma-stimulated, GAS-mediated transcription, we have termed it the IFN response-inhibitory sequence (IRIS). The combined IRIS/GAS sequence is highly conserved in mouse, rat, and bovine MCP-1 genes. In gel-shift assays, nuclear extracts from IFN-gamma-stimulated osteoblastic cells formed two specific inducible bands with labeled IRIS/GAS DNA. Both bands were supershifted by anti-STAT1 Abs, but not by Abs to STAT2, p48(ISGF-3y), IFN-regulatory factor-1, or IFN-regulatory factor-2. Formation of one of the bands required the presence of the IRIS moiety. IRIS/GAS DNA also formed a number of specific complexes with constitutively expressed factors, none of which were affected by the above Abs. These studies establish a mechanism for IFN-gamma-stimulated MCP-1 expression and identify a complex element that regulates MCP-1 gene transcription.

Base Sequence↗

PubMed1997

PU.1 is essential for p47(phox) promoter activity in myeloid cells.

Expression of the phagocyte cytosolic protein p47(phox), a component of NADPH oxidase, is restricted mainly to myeloid cells. To study the cis-elements and trans-acting factors responsible for its gene expression, we have cloned and characterized the p47(phox) promoter. A predominant transcriptional start site was identified 21 nucleotides upstream of the translation initiation codon. To identify the gene promoter sequences, transient transfections of HL-60 human myeloid cells were performed with a series of 5'-deletion p47(phox)-luciferase reporter constructs that extended as far upstream as -3050 bp relative to the transcriptional start site. The -224 and -86 constructs had the strongest p47(phox) promoter activity, whereas the -46 construct showed a major reduction in activity and the -36 construct a complete loss of activity. DNase I footprint analysis identified a protected region from -37 to -53. This region containing a consensus PU.1 site bound specifically both PU.1 present in nuclear extracts from myeloid cells and PU.1 synthesized in vitro. Mutations of this site eliminated PU.1 binding and abolished the ability of the p47(phox) promoter to direct expression of the reporter gene. The p47(phox) promoter was active in all myeloid cell lines tested (HL-60, THP-1, U937, PLB-985), but not in non-myeloid cells (HeLa, HEK293). Finally, PU.1 trans-activated the p47(phox)-luciferase constructs in HeLa cells. We conclude that, similar to certain other myeloid-specific genes, p47(phox) promoter activity in myeloid cells requires PU.1.

Animals↗

PubMed1997

Monocyte chemoattractant protein-1 mediates monocyte/macrophage influx in anti-thymocyte antibody-induced glomerulonephritis.

Chemokines are a family of chemotactic cytokines whose participation in inflammation in vivo remains to be established. To study the role of monocyte-chemoattractant-protein-1 (MCP-1) on the glomerular accumulation of leukocytes, rats received a neutralizing anti-MCP-1 antiserum following the induction of an glomerulonephritis by an anti-thymocyte antibody (ATS). The infiltration of monocytes/macrophages (M/M) and granulocytes was analyzed by immunohistology. When studied by Northern blotting, glomerular mRNA levels of MCP-1, and interleukin 1 beta (IL-1 beta) increased at three hours and 24 hours following the induction of the injury. The glomerular mRNA expression of intercellular adhesion molecule-1 (ICAM-1) only increased marginally, whereas the expression of the chemokine RANTES was not enhanced. In animals that received anti-MCP-1 antibody glomerular MCP-1 mRNA expression increased. However, the chemoattractant activity for monocytes released into supernatants of isolated glomeruli was reduced. The anti-MCP-1 antibody did not affect glomerular IL-1 beta, ICAM-1 or RANTES mRNA levels. The induction of glomerulonephritis was associated with an increased glomerular recruitment of polymorphonuclear granulocytes (PMNs) at three hours and M/M at 24 hours, when compared with controls. The anti-MCP-1 antiserum significantly reduced the glomerular M/M infiltration at 24 hours by 40%, but was without effect on glomerular PMN recruitment or growth of the resident glomerular cells. These studies demonstrate that MCP-1 is an important mediator for monocyte recruitment in this model of glomerulonephritis. The reduction of M/M infiltration might affect this glomerular injury.

Animals↗

PubMed1995

Thrombin regulates expression of monocyte chemoattractant protein-1 in vascular smooth muscle cells.

Thrombin, a serine protease generated at sites of vascular injury, plays a role in the pathogenesis of atherosclerosis and restenosis after angioplasty. Adherence of monocytes to the endothelium and migration into the subendothelial space is an important early event in the pathogenesis of atherosclerosis. Monocyte chemoattractant protein 1 (MCP-1) may be an important mediator of monocyte recruitment to the tissue in this and other diseases. We have characterized the expression of MCP-1 in vascular smooth muscle cells (VSMCs) isolated from human renal artery and studied its regulation by thrombin. Serum-deprived cells release monocyte chemotactic activity that is neutralized (80%) by an MCP-1 antibody. The antibody recognized a 13- and 15-kD protein in smooth muscle cell-conditioned medium. Thrombin stimulates MCP-1 gene expression in a concentration- and time-dependent manner. An increase over basal levels was observed with concentrations of thrombin as low as 0.05 U/mL. The maximal effect occurred at 5 U/mL. The stimulatory effect was detected within 1 hour, reached a maximum at 3 hours, and was still present at 8 to 24 hours after the addition of thrombin. A concentration- and time-dependent effect of thrombin on MCP-1 gene expression was also found in rat VSMCs. The thrombin protease inhibitor hirudin blocked thrombin-induced MCP-1 expression. Thrombin stimulated the release of MCP-1 protein in conditioned medium of human VSMCs as measured by radioimmunoassay and chemotactic assay. Thrombin also increased monocyte chemotactic activity in short-term organ cultures of rat aortic rings and in first passage cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

Thrombin stimulates proliferation of liver fat-storing cells and expression of monocyte chemotactic protein-1: potential role in liver injury.

Liver fat-storing cells (FSC) proliferate and secrete extracellular matrix in experimental models of liver injury. In this study, we determined if thrombin, a serine protease produced during acute and chronic tissue injury, modulates the functions of FSC. Thrombin stimulated DNA synthesis and proliferation of FSC, as assessed by [3H]-thymidine incorporation assay and measurement of cell number, respectively. Thrombin also increased the secretion of monocyte chemotactic protein-1 (MCP-1) in a time- and dose-dependent fashion. The effect of thrombin on both DNA synthesis and MCP-1 secretion was neutralized by pretreatment of thrombin with hirudin. The increased MCP-1 secretion was associated with increased steady-state levels of MCP-1 messenger RNA. Pretreatment of FSC with 5 mumol/retinol for 48 hours inhibited the mitogenic effects of thrombin but not the induction of MCP-1 secretion. FSC express specific transcripts encoding for the human thrombin receptor, as shown by Northern blot analysis of poly(A)+ RNA. Proteolytic activation of the thrombin receptor results in the formation of a new N-terminus that functions as a tethered ligand. We studied the effects of a thrombin receptor activating peptide (TRAP) corresponding to the newly formed N-terminus on FSC. TRAP mimicked the effects of thrombin on [3H]-thymidine incorporation, MCP-1 secretion, and MCP-1 gene expression. This study suggest that thrombin may be involved in modulating FSC proliferation and monocyte chemotaxis during human liver disease, through proteolytic activation of its receptor.

Adipocytes↗

PubMed1994

A novel biologic activity of thrombin: stimulation of monocyte chemotactic protein production.

Thrombin is a serine protease that is released at sites of vascular injury and exerts a variety of biologic effects on different cell types. Thrombin is postulated to play a role in the pathogenesis of a number of diseases including atherosclerosis, since it activates vascular smooth muscle and endothelial cells. Thrombin mediates these effects through a specific receptor that is upregulated in vascular cells in atherosclerosis. Atherosclerosis and glomerulosclerosis are characterized by the presence of monocyte-macrophages in the lesions. Monocyte chemotactic protein (MCP-1) is believed to be an important mediator of monocyte recruitment to the tissue and can be induced in a broad variety of cells including mesangial cells. We studied the effect of thrombin on MCP-1 production and gene expression in well-characterized human mesangial cells, vascular pericytes that play a central role in fibrosis of the glomerular microvascular bed. alpha thrombin stimulates MCP-1 production and gene expression in mesangial cells in a dose- and time-dependent manner. Experiments with diisopropylfluorophosphate thrombin and gamma thrombin demonstrate that this thrombin effect requires both receptor binding as well as catalytic activity, features consistent with the known properties of the recently characterized and cloned thrombin receptor. Moreover, a human thrombin receptor activating peptide (TRAP1-7) also stimulates MCP-1 production. Northern blot analysis demonstrated that mesangial cells express an mRNA transcript that hybridizes with labeled human thrombin receptor cDNA. These data describe a novel biologic activity of thrombin and suggest an additional mechanism by which this coagulation factor may participate in the progression of glomerulosclerosis, and by analogy, atherosclerosis.

Amino Acid Sequence↗

PubMed1994

Expression of monocyte chemoattractant protein 1 in human osteoblastic cells stimulated by proinflammatory mediators.

Monocyte chemoattractant protein 1 (MCP-1) is a member of the chemokine superfamily of genes that induces chemotaxis of monocytes in inflammatory processes. The effects of interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), transforming growth factor beta (TGF-beta), platelet-derived growth factor (PDGF-BB), parathyroid hormone (PTH), and 1,25(OH)2D3 on MCP-1 expression in human osteoblastic cells were compared. Inflammatory or proinflammatory cytokines stimulated the production of MCP-1 in normal human osteoblastic cells as determined by RIA. The osteotrophic mediators PTH and 1,25(OH)2D3 and PDGF-BB had no effect on MCP-1 expression. In further studies, the steady-state mRNA and MCP-1 protein levels in two human osteoblastic cell lines, MG-63 and SaOS-2, were examined. MCP-1 expression at both the protein and mRNA levels was greatly increased by IL-1 beta and TNF-alpha. At the mRNA level, IL-1 beta and TNF-alpha strongly induced MCP-1 expression; TGF-beta and IL-6 induced MCP-1 but to a lesser extent. No significant changes in MCP-1 mRNA or MCP-1 protein secretion were observed when cells were treated with PDGF-BB, PTH, and 1,25(OH)2D3. When tested on preosteoclasts, MCP-1 was shown to have no effect on the formation of multinucleated, tartrate-resistant acid phosphatase (TRAP)-positive osteoclastic cells.

Autoradiography↗

PubMed1994

Prevention of atherosclerosis and end-organ damage: a basis for antihypertensive interventional strategies.

AIM: To review the nature of the complex relationships between essential hypertension and cardiovascular end-organ damage, with a particular focus on the pathogenesis and prevention of coronary heart disease, the major complication of untreated hypertension. RISK FACTORS FOR CORONARY HEART DISEASE: Both atherosclerosis and hypertension have their origins in childhood; in the second and third decades of life development of the more advanced fibrous plaques accelerates, emphasizing the need for early diagnosis and intervention. Perplexing and complex relationships have been found among the principal risk factors for coronary heart disease, hyperinsulinemia, insulin resistance, dyslipidemia and hypertension. In the pathogenesis of atherosclerosis at the cellular and molecular level, the important features are the effects of monocyte-macrophages, oxidant stress, lipoprotein modification, inflammatory mediators and the focal hemodynamic environment. Even brief periods of experimental hypertension can accentuate atherogenesis, the effects of which are greatest but not limited to the cervical and cerebral arteries. Further, acute hypertension lasting for even a few minutes causes a 'leakage' of plasma proteins and particulate probes into the artery wall, which has far-reaching implications for antihypertensive therapy. Recent work has shown that 24-h blood pressure variability is correlated with target-organ damage in hypertensive patients. THERAPY: Antihypertensive therapy should not only lower blood pressure but also prevent significant short-term blood pressure fluctuations. The trough: peak ratio has been used to assess the effect of antihypertensive treatment on blood pressure variability. CONCLUSION: More intensive research is required to clarify the nature of the interface between hypertension and atherogenesis.

Adolescent↗

PubMed1994

Gamma interferon stimulates monocyte chemotactic protein (MCP-1) in human mesangial cells.

Cell-mediated immunity and monocyte infiltration is a prominent histologic feature of several different types of glomerulonephritis. Monocyte influx to the glomerulus correlates with glomerular hypercellularity and proteinuria. Glomerular mesangial cells, in addition to being targets for inflammatory stimuli, are also effector cells that actively participate in glomerular pathology. Mesangial cells release monocyte chemotactic protein (MCP-1). In the present article, we characterized and studied the regulation of MCP-1 released by cultured human mesangial cells. Serum-deprived mesangial cells constitutively release chemotactic activity that is neutralized by specific anti-MCP-1 antibody. An antibody to baboon MCP-1 recognized 16, 15, and 11 kd proteins from concentrated conditioned medium that were consistent with the presence of different forms of MCP-1. Gamma interferon (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1 (IL-1) markedly stimulate the release of MCP-1 as measured by a specific and sensitive radioimmunoassay. The release of MCP-1 in response to these cytokines is at least partially dependent on de novo synthesis of the protein because all three cytokines markedly stimulate the expression of MCP-1 mRNA. These data demonstrate that human mesangial cells synthesize and release at least three different forms of MCP-1 and that IFN-gamma and other cytokines regulate the secretion of MCP-1. IFN-gamma and MCP-1 may play a major role in the recruitment and activation of monocytes to the inflamed glomerulus.

Blotting, Northern↗

PubMed1993

The expression of monocyte chemotactic protein (MCP-1) in human vascular endothelium in vitro and in vivo.

A monocyte chemotactic protein (MCP-1) is thought to play a major role in recruiting monocytes to the vascular endothelium where the adherence of monocytes is one of the earliest events in atherogenesis. We cloned MCP-1 cDNA from a lambda gt 11 cDNA library constructed from human aortic endothelial mRNA to test whether MCP-1 expressed in arterial endothelium is identical to those from other sources. A approximately 670 bp MCP-1 cDNA clone was identified and showed the identical sequence with the ones from other cell lines. Northern blot analysis using this cloned MCP-1 cDNA as probe revealed two hybridizing bands of RNA at 0.68 and 0.77 kb in human aortic, human pulmonary arterial, and human umbilical vein endothelial cell cultures. Primer extension analysis showed that the difference in size (approximately 90 bp) between the two transcripts is not due to a difference at the 5'-noncoding region. The amount of MCP-1 transcripts increased dramatically in aortic endothelial cells when stimulated with recombinant IL-1 alpha (100 units/ml), IL-1 beta (100 units/ml), or TNF-alpha (200 ng/ml). Northern blot and slot blot analysis of RNA isolated from both the endothelium and the underlying vessel wall of freshly removed human arteries and veins showed MCP-1 transcripts. This observation demonstrates for the first time that MCP-1 is expressed not only in atherosclerotic human arteries but also in symptom free arteries and veins in vivo.

Amino Acid Sequence↗

PubMed1993

A modern view of atherogenesis.

Two key events in the atherogenic cascade are the focal influx and accumulation of low-density lipoprotein (LDL) cholesterol at arterial sites having a predilection for atherosclerotic lesion development and the recruitment of blood monocytes to these lesion-prone sites. Both processes are enhanced in the setting of hyperlipidemia and dyslipoproteinemia. The monocytes recruited to the endothelial surface subsequently migrate to the subendothelial space under the directed guidance of chemoattractants, such as monocyte chemotactic protein-1 and oxidatively modified LDL. These cells then undergo activation-differentiation to become macrophages. At the same time, LDL, and probably other lipoproteins such as the small dense LDL particles and lipoprotein (a), traverse the endothelium and undergo oxidative modification by reactive oxygen species. These oxidatively modified lipoproteins are recognizable by the non-down-regulating macrophage scavenger receptor. Their uptake by these receptors results in the formation of the foam cell characteristic of early-stage atherosclerosis. As monocyte recruitment and lipoprotein influx continue, the lesion grows and develops into the fatty streak. Subsequent foam cell necrosis due to the influence of cytotoxic oxidatively modified LDL and increased collagen synthesis by intimal smooth muscle cells lead to the established atherosclerotic lesion referred to as the fibrous plaque. As our understanding of the mechanisms involved in the pathogenesis of atherosclerosis has evolved over the past few years, novel strategies for intervention in the atherogenic process have emerged.

Animals↗