Constance J. Temm

Preservation of peritubular capillary endothelial integrity and increasing pericytes may be critical to recovery from postischemic acute kidney injury

Decreased renal blood flow following an ischemic insult contributes to a reduction in glomerular filtration. However, little is known about the underlying cellular or subcellular mechanisms mediating reduced renal blood flow in human ischemic acute kidney injury (AKI) or acute renal failure (ARF). To examine renal vascular injury following ischemia, intraoperative graft biopsies were performed after reperfusion in 21 cadaveric renal allografts. Confocal fluorescence microscopy was utilized to examine vascular smooth muscle and endothelial cell integrity as well as peritubular interstitial pericytes in the biopsies. The reperfused, transplanted kidneys exhibited postischemic injury to the renal vasculature, as demonstrated by disorganization/disarray of the actin cytoskeleton in vascular smooth muscle cells and disappearance of von Willebrand factor from vascular endothelial cells. Damage to peritubular capillary endothelial cells was more severe in subjects destined to have sustained ARF than in those with rapid recovery of their graft function. In addition, peritubular pericytes/myofibroblasts were more pronounced in recipients destined to recover than those with sustained ARF. Taken together, these data suggest damage to the renal vasculature occurs after ischemia-reperfusion in human kidneys. Preservation of peritubular capillary endothelial integrity and increasing pericytes may be critical to recovery from postischemic AKI.

LOX-1 and inflammation: a new mechanism for renal injury in obesity and diabetes

The early nephropathy in obese, diabetic, dyslipidemic (ZS) rats is characterized by tubular lipid accumulation and pervasive inflammation, two critically interrelated events. We now tested the hypothesis that proximal tubules from ZS obese diabetic rats in vivo, and proximal tubule cells (NRK52E) exposed to oxidized LDL (oxLDL) in vitro, change their normally quiescent epithelial phenotype into a proinflammatory phenotype. Urine of obese diabetic rats contained more lipid peroxides, and LOX-1, a membrane receptor that internalizes oxidized lipids, was mobilized to luminal sites. Levels of ICAM-1 and focal adhesion kinase, which participate in leukocyte migration and epithelial dedifferentiation, respectively, were also upregulated in tubules. NRK52E cells exposed to oxLDL showed similar modifications, plus suppression of anti-inflammatory transcription factor peroxisome proliferator-activated receptor-delta. In addition, oxLDL impaired epithelial barrier function. These alterations were prevented by an anti-LOX-1 antibody. The data support the concept that tubular LOX-1 activation driven by lipid oxidants in the preurine fluid is critical in the inflammatory changes. We suggest that luminal lipid oxidants and abnormal tubular permeability may be partly responsible for the renal tubulointerstitial injury of obesity, diabetes, and dyslipidemia.

Anandamide decreases glomerular filtration rate through predominant vasodilation of efferent arterioles in rat kidneys

For determining the effects of anandamide (ANA) on renal hemodynamics and microcirculation, a clearance study was performed in Sprague-Dawley rats that received injections of ANA in doses of 15, 150, and 1500 pmol/kg. At doses up to 150 pmol/g, ANA significantly decreased GFR and increased renal blood flow (RBF) without affecting mean arterial pressure (MAP). In the presence of the cannabinoid type 1 (CB1) receptor antagonist AM251, only the 15-pmol/kg dose significantly increased GFR and RBF without altering MAP, with higher doses having no effect on GFR, RBF, or MAP. By contrast, AM281, which antagonizes cannabinoid receptors nonselectively, inhibited the GFR, RBF, and MAP responses to ANA. The arteriolar responses to ANA were also assessed in vitro by the blood-perfused juxtamedullary nephron technique. Higher doses of ANA significantly increased the diameter of both afferent and efferent arterioles, whereas lower doses elicited predominant efferent arteriolar dilation. AM251 attenuated the afferent arteriolar response to ANA and inhibited the efferent arteriolar response to ANA, whereas AM281 inhibited the responses in both arterioles. The CB1 receptor mRNA was expressed in afferent arterioles, and immunohistochemical staining demonstrated the presence of CB1 receptors in both afferent and efferent arterioles. These results suggest that ANA causes afferent arteriolar dilation via both CB1 and non-CB1 receptors and greater efferent arteriolar dilation via CB1 receptors, resulting in a decreased GFR and an increased RBF without affecting MAP.

Differences in the temporal expression of regulatory growth factors during choroidal neovascular development

Although the roles of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and hepatocyte growth factor (HGF) in pathologic neovascularization have been well characterized in certain tissues, their particular functions and expression patterns in choroidal neovascularization (CNV) have not been clearly established. After localized laser trauma to Bruchs membrane to induce CNV development, the temporal changes in mRNA and protein expression of these 3 cytokines were documented and compared histologically to areas of immunofluorescence, the proliferation of endothelial cells, neovascular development, and temporal changes in vascular permeability. Changes in mRNA and protein levels of bFGF and HGF occurred quickly and reached peak expression within hours. This activity corresponded in time to intense and localized immunofluorescence for these cytokines within the choriocapillaris within laser lesion sites. During this same initial time period, mRNA upregulation of VEGF occurred, primarily within the neural retina and this expression corresponded to intense immunolabeling of Müller cells immediately adjacent to the lesion sites. By 3 days after lasering, increased VEGF(164) protein expression was measurable, whereas early neovascular development histologically corresponded to HGF and bFGF mRNA expansion into the developing choroidal neovascular membrane (CNVM). At 7 days, CNV expansion, maturation, and increased vascular permeability corresponded to peak VEGF mRNA and protein expression and to immunofluorescence of the CNVM. Differences also occurred in the expression of precursor and activated isoforms of these cytokines in the retinal pigment epithelium/choroid as compared to those in the retina. These molecular and immunocytochemical results suggest that bFGF and HGF may be important as initial regulators neovascularization in this CNV model; whereas VEGF may be important during later phases of angiogenesis and neovascular hyperpermeability.

Cytoplasmic p63 immunohistochemistry is a useful marker for muscle differentiation: an immunohistochemical and immunoelectron microscopic study

TP63, a member of the TP53 gene family, is a nuclear marker of myoepithelial cells. Antibody against p63 is frequently used to aid in the diagnosis of prostate carcinoma, as well as in the identification of myoepithelial cells in other tissues including the breast. p63 is also a marker for squamous cell carcinoma. Recently, it was found that all p53 family members are involved in regulating the process of muscle differentiation through the retinoblastoma (RB) protein. Ablation of these p53 family functions blocks the differentiation program and promotes malignant transformation by enabling cooperating oncogenes to transform myoblasts. We therefore studied p63 expression in a number of neoplasms with myogenic differentiation. Immunohistochemical staining for p63 was performed on paraffin sections from 38 rhabdomyosarcomas, five leiomyomas, five leiomyosarcomas, five rhabdomyomas, five rhabdomyomatous Wilms tumors, three normal cardiac muscles, one medullomyoblastoma, one pleuropulmonary blastoma with rhabdomyomatous differentiation, and one teratoma with prominent rhabdomyoblasts. Each case was also stained with desmin. Unlike the nuclear staining scored in myoepithelial cells, only cytoplasmic staining for p63 was considered positive. Of 38 cases of rhabdomyosarcoma, 36 showed cytoplasmic p63 staining; 24 of these showed highlighting of cross-striations superior to that of desmin. In addition, 5/5 rhabdomyomas, 5/5 rhabdomyomatous Wilms tumors, 1/1 pleuropulmonary blastoma with rhabdomyomatous differentiation, 1/1 teratoma with atypical rhabdoblasts, and 1/1 medullomyoblastoma exhibited cytoplasmic p63 staining. Normal cardiac muscle samples (3/3) also demonstrated positive cytoplasmic staining and distinct cross-striations. Smooth muscle tumors exhibited only very focal and faint cytoplasmic staining in 5/5 leiomyomas and 4/5 leiomyosarcomas. Immunoelectron microscopic study of skeletal muscle showed p63 localization to the Z bands of sarcomeres. We conclude that p63 immunostain is a sensitive marker for skeletal muscle differentiation and highlights the cross-striations of strap cells with exceptional definition.

Critical role of phosphorylation of serine 165 of YBX1 on the activation of NF-κB in colon cancer

Y-box binding protein 1 [YBX1] is a multifunctional protein known to facilitate many of the hallmarks of cancer. Elevated levels of YBX1 protein are highly correlated with cancer progression, making it an excellent marker in cancer. The connection between YBX1 and the important nuclear factor κB [NF-κB] has never been reported. Here, we show that overexpression of wild type YBX1 [WT-YBX1] activates NF-κB, suggesting that YBX1 is a potential NF-κB activator. Furthermore, using mass spectrometry analysis we identified novel phosphorylation of serine 165 [S165] on YBX1. Overexpression of the S165A-YBX1 mutant in either HEK293 cells or colon cancer HT29 cells showed dramatically reduced NF-κB activating ability as compared with that of WT-YBX1, confirming that S165 phosphorylation is critical for the activation of NF-κB by YBX1. We also show that expression of the S165A-YBX1 mutant dramatically decreased the expression of NF-κB-inducible genes, reduced cell growth, and compromised tumorigenic ability as compared with WT-YBX1. Taken together, we provide the first evidence that YBX1 functions as a tumor promoter via NF-κB activation, and phosphorylation of S165 of YBX1 is critical for this function. Therefore, our important discovery may lead to blocking S165 phosphorylation as a potential therapeutic strategy to treat colon cancer.

Long-term spironolactone treatment reduces coronary TRPC expression, vasoconstriction, and atherosclerosis in metabolic syndrome pigs

Coronary transient receptor potential canonical (TRPC) channel expression is elevated in metabolic syndrome (MetS). However, differential contribution of TRPCs to coronary pathology in MetS is not fully elucidated. We investigated the roles of TRPC1 and TRPC6 isoforms in coronary arteries of MetS pigs and determined whether long-term treatment with a mineralocorticoid receptor inhibitor, spironolactone, attenuates coronary TRPC expression and associated dysfunctions. MetS coronary arteries exhibited significant atherosclerosis, endothelial dysfunction, and increased histamine-induced contractions. Immunohistochemical studies revealed that TRPC6 immunostaining was significantly greater in the medial layer of MetS pig coronary arteries compared to that in Lean pigs, whereas little TRPC6 immunostaining was found in atheromas. Conversely, TRPC1 immunostaining was weak in the medial layer but strong in MetS atheromas, where it was predominantly localized to macrophages. Spironolactone treatment significantly decreased coronary TRPC expression and dysfunctions in MetS pigs. In vivo targeted delivery of the dominant-negative (DN)-TRPC6 cDNA to the coronary wall reduced histamine-induced calcium transients in the MetS coronary artery medial layer, implying a role for TRPC6 in mediating calcium influx in MetS coronary smooth muscles. Monocyte adhesion was increased in Lean pig coronary arteries cultured in the presence of aldosterone; and spironolactone antagonized this effect, suggesting that coronary mineralocorticoid receptor activation may regulate macrophage infiltration. TRPC1 expression in atheroma macrophages was associated with advanced atherosclerosis, whereas medial TRPC6 upregulation correlated with increased histamine-induced calcium transients and coronary contractility. We propose that long-term spironolactone treatment may be a therapeutic strategy to decrease TRPC expression and coronary pathology associated with MetS.

A Child With Dyserythropoietic Anemia and Megakaryocyte Dysplasia Due to a Novel 5′UTR GATA1s Splice Mutation

We describe a child with dyserythropoietic anemia, thrombocytosis, functional platelet defect, and megakaryocyte dysplasia. We show that (i) this constellation of hematopoietic abnormalities was due to a germline mutation within the 5′ untranslated region (5′UTR) of globin transcription factor 1 (GATA1); (ii) the mutation impaired a 5′UTR GATA1 splicing site, with promoted production of the shortened GATA1 isoform lacking the N‐terminus; and (iii) expression of the GATA1 N‐terminus is restricted to erythroblasts and megakaryocytes in normal marrow, consistent with the patients abnormal erythropoiesis and megakaryopoiesis. Our findings provide insights into the clinically relevant in vivo function of the N‐terminal domain of GATA1 in human hematopoiesis.

BreastDefend enhances effect of tamoxifen in estrogen receptor-positive human breast cancer in vitro and in vivo

BackgroundTamoxifen (TAM) has been widely used for the treatment of estrogen receptor (ER)-positive breast cancer and its combination with other therapies is being actively investigated as a way to increase efficacy and decrease side effects. Here, we evaluate the therapeutic potential of co-treatment with TAM and BreastDefend (BD), a dietary supplement formula, in ER-positive human breast cancer.MethodsCell proliferation and apoptosis were determined in ER-positive human breast cancer cells MCF-7 by MTT assay, quantitation of cytoplasmic histone-associated DNA fragments and expression of cleaved PARP, respectively. The molecular mechanism was identified using RNA microarray analysis and western blotting. Tumor tissues from xenograft mouse model were analyzed by immunohistochemistry.ResultsOur data clearly demonstrate that a combination of 4-hydroxytamoxifen (4-OHT) with BD lead to profound inhibition of cell proliferation and induction of apoptosis in MCF-7 cells. This effect is consistent with the regulation of apoptotic and TAM resistant genes at the transcription and translation levels. Importantly, TAM and BD co-treatment significantly enhanced apoptosis, suppressed tumor growth and reduced tumor weight in a xenograft model of human ER-positive breast cancer.ConclusionBD sensitized ER-positive human breast cancer cells to 4-OHT/TAM treatment in vitro and in vivo. BreastDefend can be used in an adjuvant therapy to increase the therapeutic effect of tamoxifen in patients with ER-positive breast cancer.

Oxidized Low Density Lipoprotein (OX-LDL) Induced Arterial Muscle Contraction Signaling Mechanisms

Oxidized low-density lipoprotein cholesterol (OX-LDL), a reactive oxidant, forms when reactive oxygen spe- cies interact with LDL. Elevated OX-LDL may contribute to high blood pressure associated with diseases such as diabetes and obesity. The current study objective was to determine if OX-LDL is a vasoconstrictor acting through the OX-LDL re- ceptor (LOX1) on arterial smooth muscle and elucidate the intracellular signaling mechanism. Arteries were extracted from Sprague-Dawley rats (SD) and obese F1 offspring (ZS) of Zucker diabetic fatty rats (ZDF) x spontaneously hyper- tensive heart failure rats (SHHF). Pulmonary arterial and aortic rings and caudal arterial helical strips were attached to force transducers in muscle baths. Arterial preparations were contracted with high KCl to establish maximum force devel- opment in response to membrane depolarization (Po). Addition of OX-LDL caused contractions of varying strength de- pendent on the arterial type. OX-LDL contractions were normalized to % Po. Caudal artery was more reactive to OX-LDL than aorta or pulmonary artery. Interestingly, LOX1 density varied with arterial type in proportion to the magnitude of the contractile response to OX-LDL. OX-LDL contractions in the absence of calcium generated about 50% as much force as in normal calcium. Experiments with myosin light chain kinase and Rho kinase inhibitors, ML-9 and Y-27632, suggest OX-LDL induced contraction is mediated by additive effects of two distinct signaling pathways activated concomitantly in the presence of calcium. Results may impact development of new therapeutic agents to control hypertension associated with disorders in which circulating LDL levels are high in a high oxidizing environment.