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Transforming growth factor-beta (TGF-beta) is the prototypic member of a large family of structurally related proteins. Three vertebrate TGF-beta isoforms have been identified and termed TGF-beta1, TGF-beta2, and TGF-beta3, respectively. In addition, two receptors of the serine/threonine kinase family termed type I and II have also been identified. In this review, we focused our attention on the effects of TGF-beta on vascular development and vascular reactivity. The critical role of the TGF-beta1 and the TGF-beta type II receptor in blood vessel formation in the yolk sac has been demonstrated by gene deletion experiments. Recent investigations have also shown that isoforms of TGF-beta play a critical role in smooth muscle cell differentiation. And, finally, a role for TGF-beta1 in the regulation of vascular tone and reactivity has been suggested by studies demonstrating that TGF-beta1 can inhibit the production of potent vasodilators (such as nitric oxide) and stimulate the production of potent vasoconstrictors (such as endothelin). Taken together, these studies suggest that TGF-beta plays a critical role in blood vessel development and vascular function.

Members of the erythroid Krüppel-like factor (EKLF) multigene family contain three C-terminal zinc fingers, and they are typically expressed in a limited number of tissues. EKLF, the founding member, transactivates the beta-globin promoter by binding to the CACCC motif. EKLF is essential for expression of the beta-globin gene as demonstrated by gene deletion experiments in mice. Using a DNA probe from the zinc finger region of EKLF, we cloned a cDNA encoding a member of this family from a human vascular endothelial cell cDNA library. Sequence analysis indicated that our clone, hEZF, is the human homologue of the recently reported mouse EZF and GKLF. hEZF is a single-copy gene that maps to chromosome 9q31. By gel mobility shift analysis, purified recombinant hEZF protein bound specifically to a probe containing the CACCC core sequence. In co-transfection experiments, we found that sense but not antisense hEZF decreased the activity of a reporter plasmid containing the CACCC sequence upstream of the thymidine kinase promoter by 6-fold. In contrast, EKLF increased the activity of the reporter plasmid by 3-fold. By fusing hEZF to the DNA-binding domain of GAL4, we mapped a repression domain in hEZF to amino acids 181-388. We also found that amino acids 91-117 of hEZF confer an activation function on the GAL4 DNA-binding domain.

Abstract To identify genes involved in vascular remodeling, we applied differential mRNA display analysis to the rat carotid artery balloon injury model. One polymerase chain reaction product showing increased expression at days 2 to 14 after vascular injury was nearly identical to the mouse α 1 chain of type VIII collagen, a heterotrimeric short-chain collagen of uncertain function expressed by a limited number of cell types. By Northern analysis, expression of both chains of the type VIII collagen heterotrimer increased: collagen α 1 (VIII) mRNA expression was almost 4-fold higher than control by 7 days after vascular injury, and collagen α 2 (VIII) mRNA expression reached a maximum of almost 6-fold above baseline by 3 days after injury. By immunohistochemical analysis, type VIII collagen expression increased in the media and neointima in a localized pattern consistent with the distribution of activated dedifferentiated vascular smooth muscle cells (VSMCs). Cultured VSMCs expressed higher levels of type VIII collagen in response to serum and growth factors, notably platelet-derived growth factor (PDGF)-BB. VSMCs adhered significantly less to type VIII collagen than to type I collagen substrata and showed greater PDGF-BB–stimulated migration (by 2.2-fold) on type VIII collagen than on type I collagen. We hypothesize that increased expression of type VIII collagen by VSMCs after arterial injury may contribute to vascular remodeling through the promotion of VSMC migration.

Endotoxic shock is a life-threatening consequence of severe Gram-negative infection characterized by vascular smooth muscle cell relaxation and severe hypotension. The production of nitric oxide (NO), through the inducible NO synthase pathway, has been implicated as a major contributor in this process. We now demonstrate that heme oxygenase (HO), an enzyme that generates carbon monoxide (CO) in the course of heme metabolism, may also be involved in the hemodynamic compromise of endotoxic shock. Inducible HO (HO-1) mRNA levels are dramatically increased in aortic tissue from rats receiving endotoxin, and this increase in vascular HO-1 message is associated with an 8.9-fold increase in HO enzyme activity in vivo. Immunocytochemical staining localizes an increase in HO-1 protein within smooth muscle cells of both large (aorta) and small (arterioles) blood vessels. Furthermore, zinc protoporphyrin IX, an inhibitor of HO activity, abrogates endotoxin-induced hypotension in rats. Studies performed in rat vascular smooth muscle cells in vitro show that the induction of HO-1 mRNA is regulated at the level of gene transcription, and this induction is independent of NO production. Taken together, these studies suggest that the up-regulation of HO-1, and the subsequent production of CO, contributes to the reduction in vascular tone during endotoxic shock.

Asthma is a prevalent disease of chronic inflammation in which endogenous counterregulatory signaling pathways are dysregulated. Recent evidence suggests that innate lymphoid cells (ILCs), including natural killer (NK) cells and type 2 ILCs (ILC2s), can participate in the regulation of allergic airway responses, in particular airway mucosal inflammation. We have identified both NK cells and ILC2s in human lung and peripheral blood in healthy and asthmatic subjects. NK cells were highly activated in severe asthma, were linked to eosinophilia, and interacted with autologous eosinophils to promote their apoptosis. ILC2s generated antigen-independent interleukin-13 (IL-13) in response to the mast cell product prostaglandin D2 alone and in a synergistic manner with the airway epithelial cytokines IL-25 and IL-33. Both NK cells and ILC2s expressed the pro-resolving ALX/FPR2 receptors. Lipoxin A4, a natural pro-resolving ligand for ALX/FPR2 receptors, significantly increased NK cell-mediated eosinophil apoptosis and decreased IL-13 release by ILC2s. Together, these findings indicate that ILCs are targets for lipoxin A4 to decrease airway inflammation and mediate the catabasis of eosinophilic inflammation. Because lipoxin A4 generation is decreased in severe asthma, these findings also implicate unrestrained ILC activation in asthma pathobiology.

Despite the importance of phenotypic alterations in arterial smooth muscle cells (ASMC) during the pathogenesis of arteriosclerosis, little is known about genes that define differentiated ASMC. Using differential mRNA display, we isolated a novel gene preferentially expressed in the rat aorta and termed this gene APEG-1. The cDNA of rat APEG-1 contained an open reading frame encoding 113 amino acids, which would predict a basic protein of 12.7 kDa. The amino acid sequence of rat APEG-1 was highly conserved among human and mouse homologues (97 and 98%, respectively). Using an APEG-1 fusion protein containing an N-terminal c-Myc tag, we identified APEG-1 as a nuclear protein. By in situ hybridization, APEG-1 mRNA was expressed in rat ASMC. Although APEG-1 was expressed highly in differentiated ASMC in vivo, its expression was quickly down-regulated and disappeared in dedifferentiated ASMC in culture. In vivo, APEG-1 mRNA levels decreased by more than 80% in response to vascular injury as ASMC changed from a quiescent to a proliferative phenotype. Taken together, these data indicate that APEG-1 is a novel marker for differentiated ASMC and may have a role in regulating growth and differentiation of this cell type.

Nitric-oxide synthases (NOS) utilize L-arginine to produce NO, a potent vasodilator that contributes to the regulation of vascular tone. We demonstrated previously that transforming growth factor (TGF)-β1 down-regulates inducible NOS after its induction by interleukin (IL)-1β by decreasing the rate of inducible NOS gene transcription. In the present study we transfected reporter plasmids containing various lengths of the inducible NOS 5′-flanking region into primary cultured rat aortic smooth muscle cells and stimulated the cells with IL-1β or vehicle. IL-1β increased the activity of the plasmid containing −1485 to +31 of the inducible NOS gene by more than 10-fold, indicating the presence of IL-1β-responsive elements. Further deletion analysis revealed that a construct containing −234 to +31 of the inducible NOS gene contained the majority of promoter/enhancer activity after IL-1β stimulation. Mutation of the NF-κB site within this region partially reduced IL-1β-inducible activity; however, a large portion of activity remained independent of the NF-κB site. TGF-β1 suppressed promoter/enhancer activity after IL-1β stimulation, and this suppression was complete in the construct with a mutated NF-κB site. In addition, TGF-β1 did not decrease the binding of nuclear proteins to the NF-κB site. These data suggest that the ability of TGF-β1 to suppress inducible NOS promoter/enhancer activity occurs through a site(s) other than the NF-κB motif in vascular smooth muscle cells.

Pyrrolidinedithiocarbamate (PDTC) and N-acetylcysteine (NAC) have been used as antioxidants to prevent apoptosis in lymphocytes, neurons, and vascular endothelial cells. We report here that PDTC and NAC induce apoptosis in rat and human smooth muscle cells. In rat aortic smooth muscle cells, PDTC induced cell shrinkage, chromatin condensation, and DNA strand breaks consistent with apoptosis. In addition, overexpression of Bcl-2 suppressed vascular smooth muscle cell death caused by PDTC and NAC. The viability of rat aortic smooth muscle cells decreased within 3 h of treatment with PDTC and was reduced to 30% at 12 h. The effect of PDTC and NAC on smooth muscle cells was not species specific because PDTC and NAC both caused dose-dependent reductions in viability in rat and human aortic smooth muscle cells. In contrast, neither PDTC nor NAC reduced viability in human aortic endothelial cells. The use of antioxidants to induce apoptosis in vascular smooth muscle cells may help prevent their proliferation in arteriosclerotic lesions.

Homocysteine is an important and independent risk factor for arteriosclerosis. We showed previously that homocysteine stimulates vascular smooth muscle cell proliferation, a hallmark of arteriosclerosis. We show here that homocysteine and serum increased DNA synthesis synergistically in both human and rat aortic smooth muscle cells (RASMCs). Treatment of quiescent RASMCs with 1 mM homocysteine or 2% calf serum for 36 h increased cyclin A mRNA levels by 8- and 14-fold, respectively, whereas homocysteine plus serum increased cyclin A mRNA levels by 40-fold, indicating a synergistic induction of cyclin A mRNA. Homocysteine did not increase the half-life of cyclin A mRNA (2.9 h), but it did increase the transcriptional rate of the cyclin A gene in nuclear run-on experiments. The positive effect of homocysteine on cyclin A gene transcription was confirmed by our finding that homocysteine increased cyclin A promoter activity and ATF-binding protein levels in RASMCs. Finally, 1 mM homocysteine increased cyclin A protein levels and cyclin A-associated kinase activity by threefold. This homocysteine-induced expression lesions by promoting proliferation of vascular smooth muscle cells.

KDR/flk-1 is one of two receptors for vascular endothelial growth factor, a potent angiogenic peptide. KDR/flk-1 is an early marker for endothelial cell progenitors, and its expression is restricted to endothelial cells in vivo. To investigate the molecular mechanisms regulating expression of KDR/flk-1, we cloned and characterized the promoter of the human KDR/flk-1 gene. The transcription start site was localized by primer extension and ribonuclease protection to a nucleotide 303 base pairs (bp) 5' of the initiation methionine codon. The 5'-flanking sequence is rich in G and C residues and contains five Sp1 elements but no TATA consensus sequence. By reporter gene transfection experiments, we found that approximately 4 kilobases of KDR/flk-1 5'-flanking sequence directed high level luciferase activity in bovine aortic endothelial cells; further deletion analysis revealed positive regulatory elements between bp -225 to -164, -95 to -77, -77 to -60, and +105 to +127. Mutation of an atypical GATA sequence between bp +105 and +127 did not affect promoter activity, suggesting that GATA elements are not essential for the high level promoter activity of this gene. Consistent with endothelial cell-restricted expression of KDR/flk-1 mRNA, we found that the 4-kilobase flanking sequence directed high level promoter activity in endothelial cells but not in other cell types. To our knowledge this is the first report characterizing the KDR/flk-1 promoter. Understanding the KDR/flk-1 promoter will allow us to investigate endothelial cell-specific gene regulation and to uncover methods for targeting gene delivery specifically to endothelial cells.

Contact inhibition mediates monolayer formation and withdrawal from the cell cycle in vascular endothelial cells. In studying the cyclins--key regulators of the cell cycle--in bovine aortic endothelial cells (BAEC), we found that levels of cyclin A mRNA decreased in confluent BAEC despite the presence of 10% fetal calf serum. We then transfected into BAEC a series of plasmids containing various lengths of the human cyclin A 5' flanking sequence and the luciferase gene. Plasmids containing 3,200, 516, 406, 266, or 133 bp of the human cyclin A promoter directed high luciferase activity in growing but not confluent BAEC. In contrast, a plasmid containing 23 bp of the cyclin A promoter was associated with a 65-fold reduction in activity in growing BAEC, and the promoter activities of this plasmid were identical in both growing and confluent BAEC. Mutation of the activating transcription factor (ATF) consensus sequence at bp -80 to -73 of the cyclin A promoter decreased its activity, indicating the critical role of the ATF site. We identified by gel mobility shift analysis protein complexes that bound to the ATF site in nuclear extracts from growing but not confluent BAEC and identified (with antibodies) ATF-1 as a binding protein in nuclear extracts from growing cells. Also, ATF-1 mRNA levels decreased in confluent BAEC. Taken together, these data suggest that the ATF site and its cognate binding proteins play an important role in the downregulation of cyclin A gene expression during contact inhibition.

Background— Two opposite views of cardiac growth are currently held; one views the heart as a static organ characterized by a large number of cardiomyocytes that are present at birth and live as long as the organism, and the other views the heart a highly plastic organ in which the myocyte compartment is restored several times during the course of life. Methods and Results— The average age of cardiomyocytes, vascular endothelial cells (ECs), and fibroblasts and their turnover rates were measured by retrospective 14 C birth dating of cells in 19 normal hearts 2 to 78 years of age and in 17 explanted failing hearts 22 to 70 years of age. We report that the human heart is characterized by a significant turnover of ventricular myocytes, ECs, and fibroblasts, physiologically and pathologically. Myocyte, EC, and fibroblast renewal is very high shortly after birth, decreases during postnatal maturation, remains relatively constant in the adult organ, and increases dramatically with age. From 20 to 78 years of age, the adult human heart entirely replaces its myocyte, EC, and fibroblast compartment ≈8, ≈6, and ≈8 times, respectively. Myocyte, EC, and fibroblast regeneration is further enhanced with chronic heart failure. Conclusions— The human heart is a highly dynamic organ that retains a remarkable degree of plasticity throughout life and in the presence of chronic heart failure. However, the ability to regenerate cardiomyocytes, vascular ECs, and fibroblasts cannot prevent the manifestations of myocardial aging or oppose the negative effects of ischemic and idiopathic dilated cardiomyopathy.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a recently characterized member of the EGF family of peptide signaling factors that acts as an early response gene to growth stimuli in vascular smooth muscle cells, as well as being a potent mitogen for these cells. As many of these growth stimuli also induce a hypertrophic response in heart muscle, we examined the regulation of HB-EGF mRNA abundance and function in primary cultures of neonatal rat ventricular myocytes and adult rat ventricular myocytes (ARVM). HB-EGF mRNA levels increased 40- and 6-fold in neonatal rat ventricular myocytes and ARVM, respectively, following a 2-4-h exposure to the alpha-adrenergic agonist phenylephrine, a known hypertrophic stimulus for these cells. Phenylephrine had no effect on HB-EGF mRNA stability, and induction of HB-EGF could be blocked completely by actinomycin D. HB-EGF mRNA abundance was also increased 15-fold in ARVM maintained in defined medium that had been induced to contract at 3 Hz by continual uniform electric field stimulation, a mechanical stimulus that we have shown preserves contractile function and induces cell growth in vitro. To determine whether cardiac myocytes would respond to exogenous HB-EGF, quiescent ARVM were exposed to defined medium conditioned by transfected COS MT cells overexpressing HB-EGF. These myocytes exhibited nearly a 2-fold increase in protein content at 24 h compared with unstimulated control ARVM exposed to medium conditioned by COS cells transfected with the plasmid vector alone. Thus, neonatal and adult cardiac muscle cells respond to both neurohumoral and mechanical growth stimuli with a marked increase in HB-EGF mRNA, which may act as an early response gene to facilitate hypertrophic growth in these cells.

Induction of heparin-binding epidermal growth factor-like growth factor mRNA by protein kinase C activators. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a potent smooth muscle cell mitogen of macrophage origin. To determine whether the HB-EGF gene is transcribed and regulated in mesangial cells, we measured HB-EGF mRNA levels in cultured rat mesangial cells by RNA blot analysis. A 2.5-kb HB-EGF mRNA was detected in unstimulated mesangial cells. The protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA) increased HB-EGF mRNA levels by 15-fold in mesangial cells, and this induction of HB-EGF mRNA by TPA was both time- and dose-dependent. HB-EGF mRNA could also be stimulated by 10% fetal calf serum, ionomycin, thrombin, and endothelin-1. Staurosporine, a protein kinase C inhibitor, abolished the induction of HB-EGF mRNA by TPA and serum. To determine whether HB-EGF is mitogenic for mesangial cells, we transfected COS cells with HB-EGF expression plasmids. Culture medium from COS cells transfected with these plasmids increased 3 H-thymidine incorporation in mesangial cells in a dose-dependent manner. To our knowledge, this is the first report that HB-EGF is expressed in renal cells. This inducible transcription of HB-EGF suggests that it may have an autocrine role in mesangial cell proliferation in kidney disease.

Nitric oxide (NO), which accounts for the biologic properties of endothelium-derived relaxing factor, is synthesized from L-arginine by nitric-oxide synthase (NOS). Two classes of NOS have been identified: a constitutive, calcium-dependent isozyme (cNOS) and an inducible, calcium-independent isozyme (iNOS). NO is generated after the induction of iNOS by cytokines such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha. As a potent vasodilator, NO may have an important role in the severe hypotension of septic shock. We investigated whether dexamethasone and transforming growth factor-beta 1 (TGF-beta 1) suppress iNOS mRNA after its induction by IL-1 beta. We found that IL-1 beta induced iNOS mRNA in rat aortic smooth muscle cells (RASMC) in a dose- and time-dependent fashion. IL-1 beta promoted a dramatic and prolonged induction of RASMC iNOS mRNA that peaked at 48 h. Dexamethasone prevented this induction of iNOS mRNA only when given before the addition of IL-1 beta. In contrast, TGF-beta 1 inhibited the induction of iNOS mRNA and NO production in RASMC both before and after the addition of IL-1 beta. After 24 h of IL-1 beta stimulation, TGF-beta 1 down-regulated the iNOS mRNA that had been induced during this initial time period. In nuclear run-on experiments, we found that the down-regulation of iNOS mRNA by TGF-beta 1 in RASMC occurred at the transcriptional level. Our observation that TGF-beta 1 mediates inhibition of RASMC iNOS mRNA after its induction by cytokines may be an important insight into the treatment of septic shock.

Experiments were designed to determine whether a heterogeneity of endothelium-dependent relaxations in arteries from different vascular beds exists in experimental congestive heart failure (CHF) and to determine the mediators of those responses. CHF was produced in dogs by rapid ventricular pacing for 15 d. Rings of coronary, femoral, and renal arteries with and without endothelium from control and CHF dogs were suspended in organ chambers for measurement of isometric force. In arteries contracted with prostaglandin F2 alpha, endothelium-dependent relaxations to BHT 920 (an alpha 2-adrenergic agonist) were increased in coronary arteries from dogs with CHF (maximal relaxation: control -15 +/- 9% vs CHF -92 +/- 5%; n = 5-6; P < 0.05), with a modest enhancement in renal arteries. Relaxations to adenosine diphosphate and the calcium ionophore were unchanged. Relaxations to BHT 920 in CHF were reduced by NG monomethyl-L-arginine (L-NMMA) and pertussis toxin but not by indomethacin. These data suggest that endothelium-dependent relaxations are affected heterogeneously in CHF. The enhanced response to alpha 2-adrenergic agonists in the coronary artery is mediated by nitric oxide through a mechanism sensitive to inhibition by pertussis toxin. This selective increase in endothelium-dependent relaxations in the coronary artery may contribute to preserving coronary blood flow during CHF.

Multiple intergenic single-nucleotide polymorphisms (SNPs) near hedgehog interacting protein (HHIP) on chromosome 4q31 have been strongly associated with pulmonary function levels and moderate-to-severe chronic obstructive pulmonary disease (COPD). However, whether the effects of variants in this region are related to HHIP or another gene has not been proven. We confirmed genetic association of SNPs in the 4q31 COPD genome-wide association study (GWAS) region in a Polish cohort containing severe COPD cases and healthy smoking controls (P = 0.001 to 0.002). We found that HHIP expression at both mRNA and protein levels is reduced in COPD lung tissues. We identified a genomic region located ∼85 kb upstream of HHIP which contains a subset of associated SNPs, interacts with the HHIP promoter through a chromatin loop and functions as an HHIP enhancer. The COPD risk haplotype of two SNPs within this enhancer region (rs6537296A and rs1542725C) was associated with statistically significant reductions in HHIP promoter activity. Moreover, rs1542725 demonstrates differential binding to the transcription factor Sp3; the COPD-associated allele exhibits increased Sp3 binding, which is consistent with Sp3's usual function as a transcriptional repressor. Thus, increased Sp3 binding at a functional SNP within the chromosome 4q31 COPD GWAS locus leads to reduced HHIP expression and increased susceptibility to COPD through distal transcriptional regulation. Together, our findings reveal one mechanism through which SNPs upstream of the HHIP gene modulate the expression of HHIP and functionally implicate reduced HHIP gene expression in the pathogenesis of COPD.

Sepsis remains the leading cause of death in critically ill patients, despite modern advances in critical care. Intestinal barrier dysfunction may lead to secondary bacterial translocation and the development of the multiple organ dysfunction syndrome during sepsis. Cyclooxygenase (COX)-2 is highly upregulated in the intestine during sepsis, and we hypothesized that it may be critical in the maintenance of intestinal epithelial barrier function during peritonitis-induced polymicrobial sepsis. COX-2−/− and COX-2+/+ BALB/c mice underwent cecal ligation and puncture (CLP) or sham surgery. Mice chimeric for COX-2 were derived by bone marrow transplantation and underwent CLP. C2BBe1 cells, an intestinal epithelial cell line, were treated with the COX-2 inhibitor NS-398, PGD2, or vehicle and stimulated with cytokines. COX-2−/− mice developed exaggerated bacteremia and increased mortality compared with COX-2+/+ mice following CLP. Mice chimeric for COX-2 exhibited the recipient phenotype, suggesting that epithelial COX-2 expression in the ileum attenuates bacteremia following CLP. Absence of COX-2 significantly increased epithelial permeability of the ileum and reduced expression of the tight junction proteins zonula occludens-1, occludin, and claudin-1 in the ileum following CLP. Furthermore, PGD2 attenuated cytokine-induced hyperpermeability and zonula occludens-1 downregulation in NS-398–treated C2BBe1 cells. Our findings reveal that absence of COX-2 is associated with enhanced intestinal epithelial permeability and leads to exaggerated bacterial translocation and increased mortality during peritonitis-induced sepsis. Taken together, our results suggest that epithelial expression of COX-2 in the ileum is a critical modulator of tight junction protein expression and intestinal barrier function during sepsis.

BACKGROUND Atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) are a family of structurally related peptides that participate in the integrated control of renal and cardiovascular function. Previous studies suggest a functional role for these hormonal peptides in cardiorenal regulation in congestive heart failure (CHF). METHODS AND RESULTS The present studies were performed in normal subjects (n = 6) and in patients with mild (New York Heart Association [NYHA] class I to II, n = 20) and severe (NYHA class III to IV, n = 20) CHF by use of radioimmunoassay and immunohistochemical staining (IHCS). Plasma ANP was significantly increased in both mild and severe CHF compared with normal subjects. In contrast, plasma BNP was only moderately increased in the severe CHF group, and plasma CNP concentration was unchanged in CHF compared with normal subjects. Atrial tissue concentrations of the natriuretic peptides did not parallel circulating concentrations. ANP predominated in normal atrial tissue, but BNP predominated in CHF. In ventricular tissue, IHCS staining was present for all three peptides in normal ventricular myocardium and was markedly enhanced in CHF. CONCLUSIONS These studies support a differential regulation of ANP, BNP, and CNP circulating concentrations and tissue activity in human CHF.

Atrial natriuretic factor (ANF) is a circulating 28-amino acid peptide that functions in the regulation of sodium homeostasis and vascular tone. ANF metabolism occurs via degradation by neutral endopeptidase 24.11 and binding to the ANF clearance receptor (ANFR-C). The present study was performed on anesthetized dogs, normal (control) and with experimental congestive heart failure (CHF), and was designed to investigate the ability of an ANF ligand specific for ANFR-C [C-ANF-(4-23)] to increase plasma ANF and also to evaluate the influence of ANFR-C on regional pulmonary and renal ANF clearances. C-ANF-(4-23) increased plasma ANF in controls (51 +/- 15 to 123 +/- 39 pg/ml; P < 0.05) and further increased plasma ANF in CHF dogs (242 +/- 30 to 327 +/- 34; P < 0.05), demonstrating that ANFR-C plays a significant role in the overall metabolism and clearance of ANF, even with chronically elevated ANF. Infusion of C-ANF-(4-23) produced a marked decrease in ANF pulmonary clearance (PCLANF) in controls (1,018 +/- 405 to -286 +/- 383 ml/min; P < 0.05); however, PCLANF was not altered by the ANF ligand in CHF dogs [-137 +/- 174 to -106 +/- 226 ml/min; not significant (NS)], suggesting an occupancy of ANFR-C or a downregulation of this receptor with chronically elevated plasma ANF. ANF renal clearance (RCLANF) was not altered in either group by C-ANF-(4-23) infusion.(ABSTRACT TRUNCATED AT 250 WORDS)

SPECIFIC AIMThe present study was designed to elucidate a role for the adipocyte fatty acid binding protein AFABP or aP2 in the development of atherosclerosis. We evaluated the expression of aP2 in atherosclerotic lesions, assessed aP2 gene regulation in macrophages, and intercrossed aP2−/− mice with apolipoprotein E-deficient (ApoE−/−) mice to study the effect of aP2 deficiency on lesion formation in this hypercholesterolemic model of atherosclerosis.PRINCIPAL FINDINGS1. aP2 is expressed in atherosclerotic lesions from ApoE−/− miceLesions from ApoE−/− mice were evaluated because they developed severe hypercholesterolemia and atherosclerotic lesions characteristic of human disease. aP2 mRNA was not detectable in the arterial walls of wild-type mice except in adipose tissue of the adventitia. However, an intense aP2 signal was visible in the arterial walls of ApoE−/− mice. The signal for aP2 was prominent in both the atherosclerotic lesion and adipose tissue of the adventitia, with much less signal in the ...

BackgroundThe architectural transcription factor High Mobility Group-A1 (HMGA1) binds to the minor groove of AT-rich DNA and forms transcription factor complexes ("enhanceosomes") that upregulate expression of select genes within the inflammatory cascade during critical illness syndromes such as acute lung injury (ALI). AT-rich regions of DNA surround transcription factor binding sites in genes critical for the inflammatory response. Minor groove binding drugs (MGBs), such as Distamycin A (Dist A), interfere with AT-rich region DNA binding in a sequence and conformation-specific manner, and HMGA1 is one of the few transcription factors whose binding is inhibited by MGBs.ObjectivesTo determine whether MGBs exert beneficial effects during endotoxemia through attenuating tissue inflammation via interfering with HMGA1-DNA binding and modulating expression of adhesion molecules.Methodology/principal findingsAdministration of Dist A significantly decreased lung and liver inflammation during murine endotoxemia. In intravital microscopy studies, Dist A attenuated neutrophil-endothelial interactions in vivo following an inflammatory stimulus. Endotoxin induction of P-selectin expression in lung and liver tissue and promoter activity in endothelial cells was significantly reduced by Dist A, while E-selectin induction was not significantly affected. Moreover, Dist A disrupted formation of an inducible complex containing NF-kappaB that binds an AT-rich region of the P-selectin promoter. Transfection studies demonstrated a critical role for HMGA1 in facilitating cytokine and NF-kappaB induction of P-selectin promoter activity, and Dist A inhibited binding of HMGA1 to this AT-rich region of the P-selectin promoter in vivo.Conclusions/significanceWe describe a novel targeted approach in modulating lung and liver inflammation in vivo during murine endotoxemia through decreasing binding of HMGA1 to a distinct AT-rich region of the P-selectin promoter. These studies highlight the ability of MGBs to function as molecular tools for dissecting transcriptional mechanisms in vivo and suggest alternative treatment approaches for critical illness.

Heme oxygenase (HO)-1 is a cytoprotective enzyme with anti-inflammatory properties. HO-1 is induced during a systemic inflammatory response, and expression of HO-1 is beneficial during sepsis of a Gram-positive source. Systemic infection from Gram-positive organisms has emerged as an important cause of sepsis, with Staphylococcus aureus as a common etiology. An important mediator of Gram-positive infections is peptidoglycan (PGN), a cell wall component of these organisms. Here, we demonstrate that HO-1 played an important, protective role in vivo, as mice deficient in HO-1 were very sensitive to the lethal effects of PGN derived from S. aureus. PGN induced HO-1 protein and mRNA levels, and this regulation occurred at the level of gene transcription. The PGN-responsive region of the HO-1 promoter (from −117 to −66 bp) contains a functional EBS, and Ets proteins are known to be involved in the regulation of inflammatory responses. We showed previously that Ets factors (activators Ets-2 and Ets-1 and repressor Elk-3) regulate HO-1 expression by Gram-negative endotoxin. However, during exposure to a Gram-positive stimulus in the present study, Elk-1 was a potent activator of HO-1 in conjunction with PGN. The ability of Elk-1 to induce HO-1 promoter activity was independent of direct DNA binding, but rather occurred by interacting with the CCAAT/enhancer-binding protein-α (C/EBPα), which binds to DNA. Moreover, silencing of C/EBPα in macrophages prevented induction of HO-1 promoter activity by either Elk-1 or PGN. These data provide further insight into the regulation and function of HO-1 by a mediator of Gram-positive bacteria.

To determine whether the gene encoding the recently identified heparin-binding epidermal growth factor-like growth factor (HB-EGF), a potent smooth muscle cell (SMC) mitogen of macrophage origin, is transcribed and regulated in vascular SMC, we isolated cDNA clones encoding rat HB-EGF from a macrophage library. Using the rat HB-EGF cDNA as a probe for RNA blot analysis, we detected low levels of HB-EGF mRNA in rat aortic SMC in culture. However, 20 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) and 10(-6) M angiotensin II (AII) induced a marked increase in HB-EGF mRNA levels in rat aortic SMC (11- and 4.6-fold, respectively) that was both dose- and time-dependent. In response to TPA and AII, HB-EGF mRNA levels increased rapidly, peaked at 2 h, and returned to base line at 7 h. This effect of AII on HB-EGF induction was specific, as evidenced by the fact that it could be completely blocked by the AII antagonist saralasin. This is the first demonstration that HB-EGF is transcribed and regulated in SMC. The inducible transcription of this potent SMC mitogen gene in vascular SMC suggests that HB-EGF may have an important autocrine role in the proliferation of SMC in vascular diseases such as atherosclerosis and hypertension.

The pulmonary and renal vasculatures, in contrast to the systemic vasculature, constrict during hypoxia. The endothelium has been implicated in mediating these vascular responses to acute hypoxia via the production of endothelium-derived vasoactive factors. The present study, performed in anesthetized dogs, was designed to investigate the role of endothelium-derived relaxing factor (EDRF) to attenuate the vasoconstrictor response of the pulmonary and renal circulations during acute hypoxia. In response to hypoxia, pulmonary (2.2 +/- 0.3 to 4.5 +/- 0.6 mmHg.l-1.min) and renal (0.60 +/- 0.07 to 0.90 +/- 0.14 mmHg.ml-1.min) vascular resistances increased. Inhibition of endogenous EDRF with NG-monomethyl-L-arginine resulted in similar increases in pulmonary (3.0 +/- 0.1 to 4.8 +/- 0.4 mmHg.l-1.min) and renal (0.67 +/- 0.07 to 0.90 +/- 0.09 mmHg.ml-1.min) vascular resistances as in hypoxia. However, in the presence of both hypoxia and EDRF inhibition, an exaggerated pulmonary vascular response was observed (2.2 +/- 0.2 to 7.4 +/- 0.9 mmHg.l-1.min), in contrast to the renal vascular response to EDRF inhibition during hypoxia (0.61 +/- 0.05 to 0.95 +/- 0.10 mmHg.ml-1.min), which was not different from hypoxia or EDRF inhibition individually. The endothelium-derived contracting factor endothelin, which modestly increased during hypoxia (11.7 +/- 1.9 to 15.6 +/- 2.4 pg/ml), may also contribute to this vasoconstrictive response to hypoxia. This study suggests in the intact animal that EDRF serves to oppose the pulmonary vasoconstrictor response to hypoxia and further characterizes the role of endothelium-derived factors in the regulation of vascular function during hypoxia.

Pulmonary arterial hypertension (PAH) is a significant disease process characterized by elevated pulmonary vascular resistance leading to increased right ventricular afterload and ultimately progressing to right ventricular dysfunction and often death. Irreversible remodeling of the pulmonary vasculature is the hallmark of pulmonary hypertension and frequently leads to progressive functional decline in patients with PAH despite treatment with currently available therapies. Metabolites of the arachidonic acid cascade play an important homeostatic role in the pulmonary vasculature, and dysregulation of pathways downstream of arachidonic acid plays a central role in the pathobiology of PAH. Cyclooxygenase-2 (COX-2) is up-regulated in pulmonary artery smooth muscle cells (PASMC) and inflammatory cells during hypoxia and plays a protective role in the lung's response to hypoxia. We recently demonstrated that absence of COX-2 was detrimental in a mouse model of hypoxia-induced pulmonary hypertension. Exposure of COX-2 null mice to hypoxia resulted in severe pulmonary hypertension characterized by enhanced pulmonary vascular remodeling and significant up-regulation of the endothelin-1 receptor ET A R in the lung after hypoxia. Absence of COX-2 in vitro led to enhanced contractility of PASMC after exposure to hypoxia, which could be attenuated by iloprost, a prostaglandin I 2 analog. These findings suggest that selective inhibition of COX-2 may have detrimental pulmonary vascular consequences in patients with preexisting pulmonary hypertension or underlying hypoxemic lung diseases. Here, we discuss our recent data demonstrating the adverse consequences of COX-2 inhibition on pulmonary vascular remodeling and PASMC contractility.

The pathological hallmarks of idiopathic pulmonary fibrosis include proliferating fibroblasts and myofibroblasts, as well as excessive collagen matrix deposition. In addition, both myofibroblast contraction and remodeling of the collagen-rich matrix contribute to the abnormal structure and function of the fibrotic lung. Little is known, however, about collagen-associated proteins that promote fibroblast and myofibroblast retention, as well as the proliferation of these cells on the extracellular matrix. In this study, we demonstrate that aortic carboxypeptidase-like protein (ACLP), a collagen-associated protein with a discoidin-like domain, is expressed at high levels in human fibrotic lung tissue and human fibroblasts, and that its expression increases markedly in the lungs of bleomycin-injured mice. Importantly, ACLP-deficient mice accumulated significantly fewer myofibroblasts and less collagen in the lung after bleomycin injury, as compared with wild-type controls, despite equivalent levels of bleomycin-induced inflammation. ACLP that is secreted by lung fibroblasts was retained on fibrillar collagen, and ACLP-deficient lung fibroblasts that were cultured on collagen exhibited changes in cell spreading, proliferation, and contraction of the collagen matrix. Finally, the addition of recombinant discoidin-like domain of ACLP to cultured ACLP-deficient lung fibroblasts restored cell spreading and increased the contraction of collagen gels. Therefore, both ACLP and its discoidin-like domain may be novel targets for anti-myofibroblast-based therapies for the treatment of pulmonary fibrosis.

Endogenous atrial natriuretic factor (ANF) serves a functional role to maintain sodium homeostasis and inhibit activation of the renin–angiotensin–aldosterone system in acute congestive heart failure despite arterial hypotension. However, as heart failure progresses, maximal synthesis and release of ANF from both the atrial and ventricular myocardium may occur resulting in relative ANF deficiency. This relative deficiency of ANF results in a progressive inability to excrete sodium and antagonize the renin–angiotensin–aldosterone system. Consequently, agents that increase circulating ANF and (or) enhance its local action have potential therapeutic efficacy. Recent studies suggest that inhibitors of neutral endopeptidase 24.11, which block ANF degradation, potentiate the natriuretic action of endogenous ANF independent of systemic or renal hemodynamics. This action does not parallel increases in plasma ANF and is associated with marked increases in urinary ANF and cyclic guanosine monophosphate consistent with enhanced local action of the peptide. In addition, agents that selectively bind to biologically inactive ANF clearance receptors increase endogenous plasma ANF and promote increases in renal sodium excretion. These studies suggest a therapeutic role for neutral endopeptidase inhibition and clearance receptor blockade, while advancing our understanding of the pathophysiology of ANF in congestive heart failure.Key words: congestive heart failure, atrial natriuretic factor, cyclic guanosine monophosphate, natriuresis, renin–angiotensin–aldosterone system.

Previous studies have demonstrated that the biological actions of atrial natriuretic factor (ANF) are mediated via increases in its intracellular second messenger guanosine 3',5'-cyclic monophosphate (cGMP). Because cGMP egresses rapidly from target cells after ANF binding to particulate guanylate cyclase-linked receptors, extracellular cGMP may be a useful biological marker for the action of ANF in vivo under pathophysiological conditions. The present studies tested the hypothesis that the avid sodium retention and renal ANF resistance characteristic of chronic congestive heart failure (CHF) are associated with attenuated renal cGMP responses to ANF. We assessed the natriuretic and cGMP responses to endogenous and exogenous ANF during the evolution of CHF produced by 6 days of rapid ventricular pacing in conscious dogs (n = 6). Simultaneous measurement of plasma and urinary cGMP concentrations allowed determination of the net renal generation of cGMP, an indicator of the renal contribution to total urinary cGMP excretion. In early CHF, increased sodium excretion and renal cGMP production were observed in association with increases in plasma ANF. Exogenous ANF administration (10 micrograms/kg iv) before CHF also produced parallel increases in sodium excretion and renal cGMP production. In more advanced CHF produced by 6 days of pacing, we observed avid sodium retention in association with reversal of earlier increases in renal cGMP production despite progressive increases in circulating ANF. Natriuretic and renal cGMP responses to exogenous ANF were similarly attenuated in chronic CHF. These studies suggest that 1) renal cGMP production is a useful biological marker for the renal natriuretic action of ANF; 2) endogenous ANF contributes to the maintenance of sodium excretion in early CHF via increases in renal cGMP production; and 3) the avid sodium retention and renal ANF resistance in advanced CHF are, in part, linked to attenuated renal cGMP responses to endogenous and exogenous ANF.

Heme oxygenase (HO)-1 is a cytoprotective enzyme that plays a critical role in defending the body against oxidant-induced injury during inflammatory processes. HO catalyzes the degradation of heme to carbon monoxide (CO), biliverdin, and ferrous iron. Biliverdin is converted to bilirubin, a potent endogenous antioxidant. CO has a number of biological functions, including anti-inflammatory properties. In various models of disease, HO-1 is known to play a critical role by ameliorating the pathological consequences of injury. In many of these models, the beneficial effects of HO-1 and its products of heme catabolism are by suppressing an inflammatory response. However, when investigating diseases due to microbial infections, inhibition of the inflammatory response could disrupt the ability of the immune system to eradicate an invading pathogen. Thus, questions remain regarding the role of HO-1 in microbial host defense. This microreview will address our present understanding of HO-1 and its functional significance in a variety of microbial infections.

Heme oxygenase 1 (HO-1) is a representative mediator of antioxidants and cytoprotectants against various stress stimuli including oxidants in vascular cells. Intensive insulin treatment can delay the onset and progression of diabetic retinopathy and other vascularopathies, yet little is known about insulin regulation of anti-apoptotic and antioxidant molecules such as HO-1 in vascular cells. Intravitreous injection or in vitro addition of insulin increased HO-1 protein expression in rat retina and in cultured bovine retinal pericytes, retinal endothelial cells, and retinal pigment epithelial cells. In bovine retinal pericytes, insulin induced mRNA and protein expression of HO-1 in a time- and concentration-dependent manner. Using HO-1 promoter analysis, the luciferase reporter assay showed that induction of HO-1 expression by insulin is mediated by additional response elements in the ho-1 promoter gene, which was not responsive to antioxidants. Insulin-induced HO-1 mRNA expression through activation of PI3-kinase/Akt pathway without affecting ERK and p38 MAPK. Overexpression of an adenoviral vector of native IRS1, IRS2, and Akt dominant negative or small interfering RNA transfection of Akt1 and Akt2 targeted gene demonstrated that insulin regulated HO-1 expression via IRS1 and Akt2 pathway, selectively. Further, insulin treatment prevented H2O2-induced NF-κB and caspase-8 activation and apoptosis via the IRS1/PI3K/Akt2/HO-1 pathway in the pericytes. In conclusion, we suggest that the anti-apoptotic properties of insulin are mediated partly by increasing HO-1 expression at transcriptional level via IRS1/PI3K/Akt2 activation, a potential explanation for how insulin is retarding the progression of microvascular complications induced by diabetes.

Background— Cyclooxygenase-2 (COX-2) is upregulated in pulmonary artery smooth muscle cells (PASMCs) during hypoxia and may play a protective role in the response of the lung to hypoxia. Selective COX-2 inhibition may have detrimental pulmonary vascular consequences during hypoxia. Methods and Results— To investigate the role of COX-2 in the pulmonary vascular response to hypoxia, we subjected wild-type and COX-2–deficient mice to a model of chronic normobaric hypoxia. COX-2–null mice developed severe pulmonary hypertension with exaggerated elevation of right ventricular systolic pressure, significant right ventricular hypertrophy, and striking vascular remodeling after hypoxia. Pulmonary vascular remodeling in COX-2–deficient mice was characterized by PASMC hypertrophy but not increased proliferation. Furthermore, COX-2–deficient mice had significant upregulation of the endothelin-1 receptor (ET A ) in the lung after hypoxia. Similarly, selective pharmacological inhibition of COX-2 in wild-type mice exacerbated hypoxia-induced pulmonary hypertension and resulted in PASMC hypertrophy and increased ET A receptor expression in pulmonary arterioles. The absence of COX-2 in vascular smooth muscle cells during hypoxia in vitro augmented traction forces and enhanced contractility of an extracellular matrix. Treatment of COX-2–deficient PASMCs with iloprost, a prostaglandin I 2 analog, and prostaglandin E 2 abrogated the potent contractile response to hypoxia and restored the wild-type phenotype. Conclusions— Our findings reveal that hypoxia-induced pulmonary hypertension and vascular remodeling are exacerbated in the absence of COX-2 with enhanced ET A receptor expression and increased PASMC hypertrophy. COX-2–deficient PASMCs have a maladaptive response to hypoxia manifested by exaggerated contractility, which may be rescued by either COX-2–derived prostaglandin I 2 or prostaglandin E 2 .

Endothelin, a recently discovered peptide produced by endothelial cells, contracts vascular strips in vitro with greater potency than any previously known vasoconstrictor. Infusions of pharmacologic doses of endothelin in vivo result in a prolonged pressor response and a preferential impairment of renal hemodynamic and excretory functions. Endothelin also directly stimulates the release of aldosterone from the adrenal gland and inhibits renin release in vitro. A highly sensitive and specific radioimmunoassay has confirmed that endothelin circulates in human plasma, and increased plasma endothelin levels have been associated with various cardiovascular disease states. This review summarizes the current knowledge about the molecular biologic features and physiologic actions of endothelin and also explores the role of endothelin, through its local and systemic function, as a regulator of vascular tone in normal and pathophysiologic states.

Plasma gelsolin is a circulating actin-binding protein that serves a protective role against tissue injuries. Depletion of plasma gelsolin in systemic inflammation may contribute to adverse outcomes. We examined the role of plasma gelsolin in animal models of sepsis.Animal and laboratory experiments.Academic research laboratory.Adult male mice.Mice subjected to endotoxin or cecal ligation and puncture (CLP) were treated with exogenous plasma gelsolin or placebo.We document the depletion of plasma gelsolin (25-50% of normal) in murine models of sepsis associated with the presence of circulating actin within 6 hrs of septic challenge. Repletion of plasma gelsolin leads to solubilization of circulating actin aggregates and significantly reduces mortality in endotoxemic mice (survival rates were 88% in the gelsolin group vs. 0% in the saline group, p.001) and in CLP-challenged mice (survival rates were 30% in the gelsolin group vs. 0% in the saline group, p = .001). Plasma gelsolin repletion also shifted the cytokine profile of endotoxemic mice toward anti-inflammatory (plasma interleukin-10 levels were 205 +/- 108 pg/mL in the gelsolin group vs. 39 +/- 29 pg/mL in the saline group, p = .02).We propose that circulation of particulate actin is a marker for sepsis-induced cell injury, that plasma gelsolin has a crucial protective role in sepsis, and that gelsolin replacement represents a potential therapy for this common lethal condition.