Kazuhiro Dohi

Increased plasma concentrations of adrenomedullin correlate with relaxation of vascular tone in patients with septic shock

OBJECTIVE To investigate plasma concentrations of adrenomedullin in patients with septic shock and the potential association of these concentrations with relaxation of vascular tone. DESIGN Prospective, case series. SETTING Department of Emergency and Critical Care Medicine, Nara Medical University. PATIENTS Twelve patients who fulfilled the clinical criteria for severe sepsis or septic shock (as defined by the Members of the American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference Committee) and 13 healthy volunteers. INTERVENTIONS Arterial blood samples were obtained via a 20-gauge cannula inserted into each patients radial artery. MEASUREMENTS AND MAIN RESULTS After extraction and purification, plasma adrenomedullin was measured by radioimmunoassay. Systemic vascular resistance index, pulmonary vascular resistance, cardiac index, and stroke volume index were determined with a thermodilution catheter. The mean plasma concentration of adrenomedullin was markedly higher in patients than in controls (226.1 +/- 66.4 [SEM] vs. 5.05 +/- 0.21 fmol/mL, p < .01). Moreover, these concentrations correlated significantly with cardiac index, stroke volume index, and heart rate values, and correlated significantly with decreases in diastolic blood pressure, systemic vascular resistance index, and pulmonary vascular resistance index values. CONCLUSIONS Enhanced production of adrenomedullin in patients with septic shock may contribute to reduced vascular tone, hypotension, or both. More data are needed to clarify the role of adrenomedullin in the regulation of vascular tone in this patient population.

Ventricular Adrenomedullin Levels Correlate With the Extent of Cardiac Hypertrophy in Rats

We investigated the pathophysiological significance of adrenomedullin (AM) in the development of left ventricular hypertrophy (LVH). LVH was produced by aortic banding (AB) in rats. The left ventricular weight/body weight (LV/BW) ratio, ventricular AM peptide and mRNA levels, and hemodynamics were measured at 1, 3, 7, and 21 days after the operation. Both LV/BW ratio and ventricular AM levels showed a significant increase from 1 day after the operation in the AB rats versus the sham-operated rats. Both increased in a time-dependent manner. The ventricular AM levels correlated with the LV/BW ratio (r=0.76, P<0.01). The AM mRNA levels were highly expressed at 1 day after the operation in the AB rats but showed no difference from 3 to 21 days after the operation between the AB and sham groups. The plasma AM levels showed a peak at 1 day after the operation in both groups. Then, we treated AB rats with an angiotensin-converting enzyme inhibitor (quinapril) in 2 doses (1 and 10 mg. kg-1. d-1) for 21 days. The quinapril treatment attenuated similarly both the LV/BW ratio and the ventricular AM levels. We also assessed the effects of AM and hydralazine administration for 7 days on the LV/BW ratio and hemodynamics of AB rats. Both AM and hydralazine administration reduced the blood pressure by approximately 10% compared with the nontreated AB rats, but a reduction of the LV/BW ratio was observed only in the AM-treated group (P<0.05). These results suggest that ventricular AM levels are elevated by chronic pressure overload in a time-dependent manner concomitant with the extent of LVH and that AM may play a pathophysiological role in the development of LVH in chronic pressure overload.

Adrenomedullin production is correlated with differentiation in human leukemia cell lines and peripheral blood monocytes

We demonstrated that adrenomedullin (AM) is produced and secreted from human leukemia cell lines (THP‐1 and HL‐60) as well as peripheral blood granulocytes, lymphocytes, monocytes and monocyte‐derived macrophages. Immunoreactive AM accumulated in the culture media of THP‐1 and HL‐60 cells increased according to their differentiation into macrophage‐like cells. Retinoic acid exerted synergistic effects on AM secretion from THP‐1 and HL‐60 cells when administered with tumor necrosis factor‐α, lipopolysaccharide or 12‐O‐tetradecanoyl phorbol‐13‐acetate. AM was shown to increase the scavenger receptor activity on THP‐1 cells. Thus, monocytes/macrophages should be recognized as sources of AM, and the secreted AM may modulate the function of macrophages.

Cardiac fibroblasts are major production and target cells of adrenomedullin in the heart in vitro

OBJECTIVE Adrenomedullin (AM) is a potent vasodilator peptide. Plasma AM concentration is increased in patients with various heart diseases, and both myocytes (MCs) and non-myocytes (NMCs) secrete AM and express its receptors. These facts suggest that cardiac cells possess an autocrine/paracrine capability mediated by AM. METHODS MCs and NMCs were prepared from cardiac ventricles of neonatal rats. AM and endothelin-1 concentrations were measured by radioimmunoassays, and interleukin-6 level by a specific bioassay. Total nitrite/nitrate contents were measured with a fluorescence assay kit. RESULTS A basal secretion rate of AM from NMCs was 2.8-fold higher than that from MCs. Interleukin-1beta, tumor necrosis factor-alpha and lipopolysaccharide stimulated AM secretion from NMCs but not from MCs. AM stimulated interleukin-6 production in the presence of these cytokines or lipopolysaccharide, which was more prominent in NMCs. In the presence of interleukin-1beta, AM augmented nitric oxide synthesis 2.7-fold in NMCs, but slightly in MCs. NMCs secreted endothelin-1 at a rate nine times higher than MCs, and AM inhibited endothelin-1 secretion from NMCs. CONCLUSION This in vitro study suggests that AM in the heart is mainly produced in NMCs and exerts its effects through NMCs, especially under inflammatory conditions.

Increased Excretion of Urinary Transforming Growth Factor Beta 1 in Patients with Diabetic Nephropathy

The accumulation of extracellular matrix in the glomeruli of human and experimental models of diabetic nephropathy is associated with disease progression. Transforming growth factor beta 1 (TGF-β1), which is a multifunctional peptide growth factor, plays a key role in the synthesis of extracellular matrix protein in vitro, and the expression of TGF-β1 is elevated in human and rat diabetic nephropathy. In this study, we measured the urinary TGF-β1 excretion in 57 patients with non-insulin-dependent diabetes mellitus and in 20 healthy volunteers to examine whether the determination of urinary TGF-β1 excretion would facilitate the evaluation of the degree of mesangial expansion in patients with diabetic nephropathy. Both active and total TGF-β1 levels in 24-hour urine samples collected from patients with diabetes mellitus and normal controls were measured using an enzyme-linked immunosorbent assay. We observed a higher excretion of urinary TGF-β1 in patients with diabetes mellitus than in normal controls. In addition, the urinary TGF-β1 excretion was elevated in patients with severe mesangial expansion. These results suggest that urinary TGF-β1 may represent one parameter that can be used to evaluate the progression of diabetic nephropathy.

Immunohistochemical detection of advanced glycosylation end products within the vascular lesions and glomeruli in diabetic nephropathy

Numerous studies over the years have implicated advanced glycosylation end products (AGEs) in the pathogenesis of many of the complications of diabetes and normal aging. The recent development of specific antibodies against AGE-modified proteins has facilitated investigations on the formation and tissue distribution of AGEs. We used anti-AGE antibodies to localize AGEs within kidney specimens obtained from both diabetic and nondiabetic individuals. Immunohistochemical staining using anti-AGE antibody showed a high level of AGE accumulation in diabetic and aged vascular intima, particularly along the inner elastic layer of arteries. Positive staining also was observed within nodular and severe diffuse lesions of glomeruli as well as in hyaline deposits of arterioles. These data support a pathogenic role for advanced glycosylation in the renal complications of diabetes and aging.

Intraglomerular expression of transforming growth factor-beta 1 (TGF-β1) mRNA in patients with glomerulonephritis: quantitative analysis by competitive polymerase chain reaction

TGF‐β1 is involved in the pathogenesis of glomerular sclerosis. We studied the intraglomerular expression of TGF‐β1 mRNA in patients with glomerulonephritis using competitive polymerase chain reaction (PCR). This method is sensitive enough to quantify cDNA copies of mRNA present in small amounts of samples. Renal biopsy specimens were obtained from 42 patients with various kinds of glomerulonephritis. Ten glomeruli were dissected from renal biopsy specimens. Normal glomeruli were also obtained from the resected kidneys of eight patients with renal cell cancer. Total RNA was extracted from the glomeruli and reverse transcribed into cDNA with reverse transcriptase. To prepare samples containing identical amounts of β‐actin cDNA (8 pg), we performed competitive PCR by co‐amplifying mutant templates of β‐actin with a unique EcoRI site. Next, to measure TGF‐β1 cDNA, we performed competitive PCR by co‐amplifying mutant templates of TGF‐β1. We observed a higher glomerular expression of TGF‐β1 mRNA in cases of mesangial proliferative glomerulonephritis having a moderate increase in mesangial matrix, diabetic nephropathy and diffuse proliferative lupus nephritis, compared with normal glomeruli. Results suggest that the intraglomerular synthesis of TGF‐β1 may be involved in the progression of glomerulonephritis in humans.

Serum levels of VEGF and basic FGF in the subacute phase of myocardial infarction

We examined serial changes in serum levels of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) measured by ELISAs in 45 patients with acute myocardial infarction (AMI) who received heparin intravenously for 3 to 5 days after the onset and in 30 control subjects with an old myocardial infarction. To evaluate the effect of heparin on these serum levels, heparin was administered intravenously in 10 patients with AMI on day 21. Blood samples were obtained from all AMI patients on days 1, 2, 3, 7, 14, 21, and 28 and from 10 AMI patients before and 1 h after heparin administration. Serum VEGF level was significantly reduced after heparin administration (P<0.001). Serum samples from day 1 to 3 were therefore excluded from the subsequent analysis. Serum VEGF level in AMI patients was significantly higher on day 7 than in the control subjects (P<0.0001), and then decreased over time (P<0.0001). The serum VEGF level on day 7 was independently associated with the peak serum CK level (P<0.05). The serum bFGF level did not differ significantly between the AMI patients and the control subjects. In conclusion, the serum VEGF level may be selectively elevated during the healing process after AMI.

Polymorphisms of HLA class II genes and autoimmune responses to Ro/SS-A-La/SS-B among Japanese subjects

OBJECTIVE To investigate HLA class II allele associations with autoantibody responses to Ro/SS-A and La/SS-B among Japanese subjects. METHODS Haplotype and allele distributions, along with molecular polymorphisms, of HLA class II genes were analyzed by polymerase chain reaction-restriction fragment length polymorphism in 41 Japanese women with precipitating autoantibodies to Ro/SS-A and/or La/SS-B. RESULTS Among women with both Ro/SS-A and La/SS-B antibodies, the HLA class II haplotype DRB1*08032/DQA1*0103/DQB1*0601 and DRB1*08032 allele showed significantly increased frequencies compared with patients with anti-Ro/SS-A alone or with normal controls. All women with both anti-Ro/SS-A and anti-La/SS-B, but not those with anti-Ro/SS-A alone, carried DRB1 alleles that shared the same amino acid residues at positions 14-31 and 71 of the hypervariable regions of the DRB1 chain. All anti-Ro/SS-A positive women carried 1 or 2 alleles of DQB1*06 and DQB1*03 subtypes that shared the same amino acid residues at positions 71-77 of the DQB1 chain. HLA class II allele distributions did not differ among 3 anti-Ro/SS-A positive groups with different disease expressions, i.e., patients with systemic lupus erythematosus, patients with primary Sjögrens syndrome, and women with no apparent symptoms of rheumatic disease. CONCLUSION HLA class II allele distributions differ among anti-Ro/SS-A positive subjects according to the presence or absence of coexisting anti-La/SS-B antibodies, but not according to disease expression. Our findings suggest that different HLA class II molecules might control the development of anti-Ro/SS-A and/or anti-La/SS-B antibodies in the autoimmune response to the Ro/SS-A-La/SS-B complex.

Relationship between lupus nephritis activity and the serum level of soluble VCAM-1

We measured the serum levels of soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble E-selectin (sE-selectin) and soluble intercellular adhesion molecule-1 (sICAM-1) in 72 patients with systemic lupus erythematosus (SLE) (including patients with active nephritis) and 33 normal control subjects, to investigate the correlation between levels of adhesion molecules and disease and histological activity. Serum samples were obtained at the time of renal biopsy in 27 patients with lupus nephritis. The 27 patients were divided into groups according to the World Health Organization (WHO) class as follows: class I / II, n = 11; class III / IV, n = 13 and class V, n = 3. We also determined the activity index (AI) in these 27 renal biopsy specimens. We obtained serial measurements of the serum levels of soluble adhesion molecules in 11 patients to examine the difference between active and remission stages. The serum level of sVCAM-1, but not sE-selectin or sICAM-1, was correlated with parameters of SLE disease activity, including the SLE disease activity index score, the anti-double stranded DNA antibody titer, the C3 level, the C4 level and the CH50 level. The serum levels of sVCAM-1, sE-selectin and sICAM-1 were significantly higher in patients with SLE than in controls (P = 0.006, P = 0.0005 and P = 0.04, respectively). The serum level of sVCAM-1 was significantly higher in patients with active lupus nephritis (WHO classes III and IV) than in patients in inactive lupus nephritis (WHO classes I and II) (P = 0.0016). The sVCAM-1 level was significantly elevated in patients with an AI > 4 compared with patients with an AI < 4(P = 0.0025). The sVCAM-1 level decreased significantly during remission (P = 0.0033). The serum level of sVCAM-1 was elevated in patients with active lupus nephritis (WHO classes III and IV) and in patients with high AI scores. The serum level of sVCAM-1 was correlated with the SLE disease activity and decreased during remission. Therefore, the sVCAM-1 level may be a useful marker of lupus nephritis activity.