Interlobular hyaline arteriopathy reflects severe arteriolopathy in renal allografts.

AIM: The present study was performed to examine the clinicopathological significance of hyaline deposits in the smooth muscle of the interlobular artery (interlobular hyaline arteriopathy [IHA]) in renal allografts. METHODS: Tissue specimens that included the interlobular artery from biopsies performed from January 2012 to December 2015, as well as specimens from biopsies performed ≥1 year after living kidney transplantation were analyzed. Biopsies of recipients with new-onset diabetes mellitus after transplantation were excluded, as well as those of recipients who had undergone transplantation because of diabetic nephropathy. Arteriolopathy was evaluated using the aah score determined by the Banff 2007 classification. RESULTS: In total, 51 specimens with IHA lesions were identified among 381 biopsies obtained from 243 recipients performed ≥1 year after kidney transplantation. Among these 51 biopsies, 18 specimens had a score of aah3, 29 had a score of aah2, and four had a score of aah1. The incidence of IHA lesions was 3.6% at ≥1 to <4 years, 18.5% at ≥4 to <8 years, and 54.1% at ≥8 years. Older kidney grafts exhibited more IHA lesions. Among the biopsy specimens obtained ≥8 years after transplantation, no significant differences in the recipient or donor age, duration after transplantation, or prevalence of hypertension were observed between the IHA and non-IHA groups. The aah scores were significantly higher in the IHA group ≥8 years after transplantation as determined by the mean score test (P < 0.01). CONCLUSION: IHA in renal allografts is associated with severe arteriolopathy.

Intranuclear expression of progesterone receptors in smooth muscle cells of renovascular fibromuscular dysplasia: a pilot study.

BACKGROUND: The pathogenesis of fibromuscular dysplasia (FMD) remains poorly understood. Yet, understanding this mechanism has taken on new urgency after recent evidence indicating that FMD is not as rare as previously thought. We speculated that hormonal receptors in the walls of dysplastic renal arteries were implicated in the pathogenesis of FMD. METHODS: We undertook a pilot prospective case-control study comparing histologic findings from renal arteries that were surgically removed in 2 patient groups. The case group included 6 samples from FMD patients who underwent surgery for stenosis or aneurysm caused by FMD. The control group included 3 FMD-free patients who underwent nephrectomy for nonvascular causes. Surgical specimens were sent to the histology laboratory. FMD was defined preoperatively using conventional radiologic criteria and was confirmed by histologic examination. RESULTS: Immunohistochemical staining detected intense progesterone receptor expression in the nuclei of smooth muscle cells in FMD patients. No progesterone receptor expression was found in the FMD-free patients. Estrogen receptor expression was not noted in the 2 groups. CONCLUSIONS: This preliminary finding may suggest that progesterone plays a key role in the pathogenesis of FMD and opens the fields of genetic and therapeutic approaches.

Atherosclerotic plaque components characterization and macrophage infiltration identification by intravascular ultrasound elastography based on b-mode analysis: validation in vivo.

Intravascular ultrasound elastography (IVUSE) is a promising imaging technique for early investigation of vulnerable plaques. Compared to radiofrequency signal processing, digital B-mode analysis is simple and of higher portability. However, rare studies have been reported validating the latter technique in vivo. In this study, we developed an IVUSE computer software system involving semi-automatic border delineation and block-matching algorithm and validated the system in vivo. Seven minipigs were fed with atherogenic diet for 40 weeks. For each pig, the endothelium of one side of the renal arteries was denuded at the fifth week. With cross-correlation analysis, Lagrangian strain was calculated from two intravascular ultrasound images acquired in situ. Sixty regions of interests were selected from 35 elastograms matched well with the corresponding histological slices. Plaque types within these regions were classified as fibrous, fibro-fatty or fatty on Masson's trichrome and Oil-red O staining. Macrophage infiltration was also evaluated with immunohistology. Comparison between the mean strain value of the region of interest and the histological results revealed significant differences in strain values among different plaque types and non-diseased artery walls. The extent of macrophage infiltration was found to be correlated positively with strain values. For identification of fibro-fatty and fibrous plaques and macrophage infiltration, the system showed high sensitivity (93, 96 and 92%, respectively) and specificity (89, 76 and 66%, respectively), as revealed by receiver operating characteristic analysis. Our IVUSE system based on B-mode analysis is capable of characterizing fibrous and fibro-fatty plaques and macrophage intensity, thus holds potential for identifying vulnerable plaque.

Adenosine2A receptor vasodilation of rat preglomerular microvessels is mediated by EETs that activate the cAMP/PKA pathway.

Dilation of rat preglomerular microvessels (PGMV) by activation of adenosine A2A receptors (A2AR) is coupled to epoxyeicosatrienoic acid (EET) release. We have investigated the commonality of this signal transduction pathway, i.e., sequential inhibition of G(salpha), adenylyl cyclase, PKA, and Ca2+-activated K+ (KCa) channel activity, to the vasoactive responses to A2AR activation by a selective A2A agonist, CGS-21680, compared with those of 11,12-EET. Male Sprague-Dawley rats were anesthetized, and microdissected arcuate arteries (110-130 microm) were cannulated and pressurized to 80 mmHg. Vessels were superfused with Krebs solution containing NG-nitro-L-arginine methyl ester (L-NAME) and indomethacin and preconstricted with phenylephrine. We assessed the effect of 3-aminobenzamide (10 microM), an inhibitor of mono-ADP-ribosyltranferases, on responses to 11,12-EET (3 nM) and CGS-21680 (10 microM) and found that both were inhibited by approximately 70% (P<0.05), whereas the response to SNP (10 microM) was unaffected. Furthermore, 11,12-EET (100 nM), like cholera toxin (100 ng/ml), stimulated ADP-ribose formation in homogenates of arcuate arteries compared with control. SQ-22536 (10 microM), an inhibitor of adenylyl cyclase activity, and myristolated PKI (14-22) amide (5 microM), an inhibitor of PKA, decreased activity of 11,12-EET and CGS-21680. Incubation of 11,12-EET (3 nM-3 microM) with PGMV resulted in an increase in cAMP levels (P<0.05). The responses to both 11,12-EET and CGS-21680 were significantly reduced by superfusion of iberiotoxin (100 nM), an inhibitor of KCa channel activity. Thus in rat PGMV activation of A2AR is coupled to EET release upstream of adenylyl cyclase activation and EETs stimulate mono-ADP-ribosyltransferase, resulting in Gsalpha protein activation.

Rapamycin attenuates vascular wall inflammation and progenitor cell promoters after angioplasty.

Rapamycin combines antiproliferative and antiinflammatory properties and reduces neointima formation after angioplasty in patients. Its effect on transcriptional programs governing neointima formation has not yet been investigated. Here, we systematically analyzed the effect of rapamycin on gene expression during neointima formation in a human organ culture model. After angioplasty, renal artery segments were cultured for 21 or 56 days in absence or presence of 100 ng/ml rapamycin. Gene expression analysis of 2312 genes revealed 264 regulated genes with a peak alteration after 21 days. Many of those were associated with recruitment of blood cells and inflammatory reactions of the vessel wall. Likewise, chemokines and cytokines such as M-CSF, IL-1beta, IL-8, beta-thromboglobulin, and EMAP-II were found up-regulated in response to vessel injury. Markers indicative for a facilitated recruitment and stimulation of hematopoetic progenitor cells (HPC), including BST-1 and SDF-1, were also induced. In this setting, rapamycin suppressed the coordinated proadhesive and proinflammatory gene expression pattern next to down-regulation of genes related to metabolism, proliferation, and apoptosis. Our study shows that mechanical injury leads to induction of a proinflammatory, proadhesive gene expression pattern in the vessel wall even in absence of leukocytes. These molecular events could provide a basis for the recruitment of leukocytes and HPC. By inhibiting the expression of such genes, rapamycin may lead to a reduced recruitment of leukocytes and HPC after vascular injury, an effect that may play a decisive role for its effectiveness in reducing restenosis.

Expression of A20 in the vessel wall of rat-kidney allografts correlates with protection from transplant arteriosclerosis.

BACKGROUND: Chronic rejection with development of transplant arteriosclerosis is the major culprit involved in loss of kidney allografts. The allografts' fate was thought to depend on the intensity of the host immune responses and the potency of immunosuppressive regimens. Recent data suggests that grafts contribute to their own survival by way of up-regulation of "cytoprotective" genes. METHODS: We analyzed the expression of four cytoprotective genes, A20, Bcl-2, Bcl-x(L) and heme oxygenase (HO)-1, in three rat renal allograft models of chronic rejection: Fisher 344-Lewis (F344/Lew), Dark Agouti-Brown Norway (DA/BN), and DA-Wistar-Furth (WF). We chose these genes for their known anti-inflammatory and anti-apoptotic function in endothelial cells (EC) and the atheroprotective function of A20 in smooth muscle cells (SMC). RESULTS: Twenty-eight and 9 weeks following transplantation, F344/Lew and DA/BN transplants had stable graft function. Histopathologic analysis showed moderate tissue damage, minimal cellular infiltrates, and preserved vascular integrity correlating with high expression of A20 in SMC. Conversely, impaired allograft function in the DA/WF combination with substantial transplant arteriosclerosis was noted in 60% of the grafts correlating with absent or decreased A20 expression in EC and SMC. In all combinations, expression of HO-1, Bcl-2, and Bcl-x(L) colocalized with infiltrating cells and was not informative on the graft status. CONCLUSIONS: We demonstrate for the first time a strict correlation between A20 expression in the vessel and the absence of transplant arteriosclerosis in rat kidney-allograft models. This data is similar to data obtained in human kidney allografts and suggests that A20 may represent a novel therapeutic target for the prevention of chronic allograft rejection.

Sex-specific acute effect of estrogen on endothelium-derived contracting factor in the renal artery of hypertensive Dahl rats.

OBJECTIVE: To determine whether estrogen rapidly affects endothelium-derived contracting factor (EDCF) in the renal artery of hypertensive Dahl rats, and whether factors other than nitric oxide (NO) contribute to the effect of estrogen. DESIGN: Acute effects of estrogen on the acetylcholine-induced vasomotor responses and on prostaglandin H2/thromboxane A2 mimetic, U46619,-induced contraction were examined in isolated arterial rings. METHODS AND RESULTS: Dahl salt-sensitive male and female rats were fed an 8% NaCl diet for 4 weeks. The blood pressure increased more rapidly and to a greater extent in males than in females. Renal arterial rings were prepared for isometric tension recording. 17beta-Estradiol, but not the biologically less active stereoisomer, 17alpha-estradiol, improved the relaxation response to acetylcholine in renal arteries from females. Estrogen also rapidly decreased the contraction evoked by acetylcholine (10(-6) to approximately 10(-4) mol/l) in renal arteries from females and it was effective at a physiological concentration (10(-9) mol/l) in the presence of Nomega-nitro-l-arginine methyl ester (an NO synthase inhibitor). The estrogen receptor antagonist, ICI 182,780, abolished the effect of estrogen, whereas the cytochrome P450 inhibitor, miconazole, had no effect. The contraction induced by U46619 was also suppressed by estrogen, without any contribution from NO. Estrogen had no effect on either relaxation or contraction responses in renal arteries from males. CONCLUSION: 17beta-Estradiol antagonizes increases in vascular tone in hypertensive females by enhancing NO-dependent relaxation, and by suppressing EDCF-mediated mechanisms in an NO-independent manner.

P2X receptor immunoreactivity in different arteries from the femoral, pulmonary, cerebral, coronary and renal circulations.

The expression of the seven P2X receptor subunits (P2X1-7) in the rat vascular system was determined using subtype-selective antibodies. Arteries of different sizes (from arterioles to conduit vessels) from a range of vascular beds were used to give an overview of receptor expression. P2X1 receptor immunoreactivity was detected in the smooth muscle layer of arteries. The relative level of P2X1 receptor immunoreactivity was dependent on the size of the artery and the vascular bed; expression was highest in small and medium arteries. P2X4 receptors were detected in all arteries; once again, the relative level of expression was dependent on the size of the artery and the vascular bed. P2X5 receptor immunoreactivity was barely detectable in most arteries studied. P2X7 receptor immunoreactivity was generally punctate and associated with the outer adventitial layer. Immunoreactivity for P2X2, P2X3 and P2X6 receptors was not detected in arteries. These results demonstrate that arteries express multiple P2X receptor subunits and that there is a heterogeneity in the level of expression. The properties of artery P2X receptors correspond to homomeric P2X1 receptors, and the function of P2X4 and P2X5 receptor subunits in arteries is unclear.

Paradoxical effects of temperature on vascular tone.

Temperature may have significant influence on vascular tone in such cases as organ preservation, coronary bypass surgery, and extracorporeal circulation. The aim of this research was to study the direct effect of temperature variation on vascular tone in an attempt to elucidate the mechanisms involved. In a first series of experiments, the isometric tension of two different vessels (rat thoracic aorta and pig renal branch artery) was studied at different temperatures. To study the role of calcium in this response, a second series of experiments was performed. In this the vessels were incubated with the intracellular chelator BAPTA/AM. Further experiments were performed to test the effect of cold storage. Our results show that changes in temperature lead to different results in pig renal artery and rat aorta. A decrease in temperature induced a highly reproducible relaxation in rat aorta, whereas pig renal artery presented cooling-induced contraction. Moreover, whereas calcium depletion failed to inhibit cooling-induced relaxation in rat aorta, it did not provoke cooling-induced contraction in pig renal artery. Similar responses were obtained with cold storage and calcium depletion. We intend to demonstrate that, just as the effect of temperature variation on pig renal artery is due to a metabolic mechanism, its effect on rat aorta may be due to structural factors. This hypothesis is supported by the result of histological studies which demonstrate a higher proportion of elastin fibres in rat aorta than in pig renal artery.

Regulation of renal artery smooth muscle tone by alpha1-adrenoceptors: role of voltage-gated calcium channels and intracellular calcium stores.

The ischemia induced vasospasm of the renal arterial blood vessels mediated by alpha1-adrenoceptors is of importance for the loss of kidney function. This is based on reduced perfusion of the kidney cortex occurring in kidney transplant and organ preserving surgery. The present study considered the intracellular mechanism of the norepinephrine (NE) induced renal artery vasospasm by using swine renal artery smooth muscle ring. Norepinephrine and phenylephrine (PE) induced dose-dependent and fully reversible isometric contractions with a threshold concentration of 10 nM (n = 7) and 10 nM (n = 4), and an EC50 of 0.3 microM and 1 microM, respectively. The receptor was identified as alpha1A-subtype. The contraction was completely inhibited by verapamil (IC50 = 1.51 microM; n = 11) and diltiazem (IC50 = 9.49 microM; n = 8) and 85% by nifedipine (IC50 = 0.13 microM; n = 21). Blockade of the intracellular inositol- 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store by thapsigargin (1 microM, n = 7) or suppression of Ca2+ release from the intracellular Ca2+-sensitive Ca2+ store by ryanodine (100 microM, n = 4) inhibited the PE induced contraction by 39.5% and 47.6%, respectively. The results suggest a key role of voltage-dependent Ca2+ channels and intracellular Ca2+ stores in the alpha1A-adrenoceptor induced contraction of the renal artery.

Alterations in endothelial F-actin microfilaments in rabbit aorta in hypercholesterolemia.

The current study tests whether hypercholesterolemia influences the distribution of endothelial cell microfilaments during the initiation and growth of fatty streak-type lesions. We classified the lesions occurring over a 20-week period into four types based on the location and extent of macrophage infiltration observed microscopically. The earliest lesion was characterized by leukocytes adherent to the endothelial surface. Minimal lesions were characterized by a few cells in the subendothelium. Intermediate lesions consisted of numerous subendothelial leukocytes in a minimally raised lesion. Advanced fatty streak lesions were elevated, with several layers of leukocytes. The organization of peripheral junctional actin (the dense peripheral band) and of central endothelial cell actin microfilament bundles was studied in each of these lesions by using fluorescent microscopy. We found that in the aorta away from branch sites and in areas away from lesions, the central microfilament distribution was unaffected by hypercholesterolemia. The macrophages entered the wall without any identifiable reorganization in the microfilaments. During the accumulation of subendothelial macrophages in minimal and intermediate lesions, stress fibers were initially increased in comparison to lesion-free areas. In raised advanced lesions, the central microfilaments became thinner and disappeared. However, at flow dividers, where central stress fibers are normally prominent, endothelial cells on the surface of intermediate lesions showed a reduction in central fibers, and peripheral bands became prominent. This finding was associated with changes in cell shape from elongated to cobblestone type. Thus, actin microfilament bundles in endothelial cells underwent substantial changes in distribution during the accumulation of subendothelial macrophages, forming hypercholesterolemia-induced fatty streak-type lesions. These changes may influence endothelial substrate adhesion, permeability, or repair after injury.

Identification of platelet-derived growth factor A and B chains in human renal vascular rejection.

Platelet-derived growth factor (PDGF) exists as a dimer composed of two homologous but distinct peptides termed PDGF-A and -B chains, and may exist as AA, AB, and BB isoforms. The PDGF-B chain has been implicated as a mediator of renal vascular rejection by virtue of up-regulated expression of its receptor, PDGF beta-receptor, in affected arteries. A role for PDGF-A chain in mediating intimal proliferation has been suggested in human atherosclerosis (Rekhter MD, Gordon D: Does platelet-derived growth factor-A chain stimulate proliferation of arterial mesenchymal cells in human atherosclerotic plaques? Circ Res 1994, 75:410), but no studies of this molecule in human renal allograft injury have been reported to date. We used two polyclonal antisera to detect expression of PDGF-A chain and one monoclonal antibody to detect PDGF-B chain by immunohistochemistry in fixed, paraffin-embedded tissue from 1) normal adult kidneys, 2) a series of renal transplant biopsies chosen to emphasize features of vascular rejection, and 3) allograft nephrectomies. Immunohistochemistry was correlated with in situ hybridization on adjacent, formalin fixed tissue sections from nephrectomies utilizing riboprobes made from PDGF-A and -B chain cDNA. PDGF-A chain is widely expressed by medial smooth muscle cells of normal and rejecting renal arterial vessels of all sizes by immunohistochemistry and in situ hybridization. PDGF-A chain is also expressed by a population of smooth muscle cells (shown by double immunolabeling with an antibody to alpha-smooth muscle actin) comprising the intima in chronic vascular rejection. In arteries demonstrating acute rejection, up-regulated expression of PDGF-A chain by endothelial cells was detected by both immunohistochemistry and in situ hybridization. In contrast, PDGF-B chain was identified principally in infiltrating monocytes within the rejecting arteries, similar to its localization in infiltrating monocytes in human atherosclerosis. Although less prominent than the case for PDGF-A chain, PDGF-B chain also was present in medial and intimal smooth muscle cells in both rejecting and nonrejecting renal arteries. PDGF-A and -B chains have now been localized at both the mRNA and protein levels to the intimal proliferative lesions of vascular rejection. These peptides, which are known stimuli for smooth muscle cell migration and proliferation in experimental vascular injury, may have similar stimulatory effects on smooth muscle cells in an autocrine and/or paracrine manner to promote further intimal expansion and lesion progression in this form of human vasculopathy.