Endothelial-Mesenchymal Transition of Brain Endothelial Cells: Possible Role during Metastatic Extravasation

Cancer progression towards metastasis follows a defined sequence of events described as the metastatic cascade. For extravasation and transendothelial migration metastatic cells interact first with endothelial cells. Yet the role of endothelial cells during the process of metastasis formation and extravasation is still unclear, and the interaction between metastatic and endothelial cells during transendothelial migration is poorly understood. Since tumor cells are well known to express TGF-beta, and the compact endothelial layer undergoes a series of changes during metastatic extravasation (cell contact disruption, cytoskeletal reorganization, enhanced contractility), we hypothesized that an EndMT may be necessary for metastatic extravasation. We demonstrate that primary cultured rat brain endothelial cells (BEC) undergo EndMT upon TGF-beta 1 treatment, characterized by the loss of tight and adherens junction proteins, expression of fibronectin, beta 1-integrin, calponin and a-smooth muscle actin (SMA). B16/F10 cell line conditioned and activated medium (ACM) had similar effects: claudin-5 down-regulation, fibronectin and SMA expression. Inhibition of TGF-beta signaling during B16/F10 ACM stimulation using SB-431542 maintained claudin-5 levels and mitigated fibronectin and SMA expression. B16/F10 ACM stimulation of BECs led to phosphorylation of Smad2 and Smad3. SB-431542 prevented SMA up-regulation upon stimulation of BECs with A2058, MCF-7 and MDA-MB231 ACM as well. Moreover, B16/F10 ACM caused a reduction in trans-endothelial electrical resistance, enhanced the number of melanoma cells adhering to and transmigrating through the endothelial layer, in a TGF-beta-dependent manner. These effects were not confined to BECs: HUVECs showed TGF-beta-dependent SMA expression when stimulated with breast cancer cell line ACM. Our results indicate that an EndMT may be necessary for metastatic transendothelial migration, and this transition may be one of the potential mechanisms occurring during the complex phenomenon known as metastatic extravasation

LPS-induced delayed preconditioning is mediated by hsp90 and involves the heat shock response in mouse kidney.

INTRODUCTION: We and others demonstrated previously that preconditioning with endotoxin (LPS) protected from a subsequent lethal LPS challenge or from renal ischemia-reperfusion injury (IRI). LPS is effective in evoking the heat shock response, an ancient and essential cellular defense mechanism, which plays a role in resistance to, and recovery from diseases. Here, by using the pharmacological Hsp90 inhibitor novobiocin (NB), we investigated the role of Hsp90 and the heat shock response in LPS-induced delayed renal preconditioning. METHODS: Male C57BL/6 mice were treated with preconditioning (P: 2 mg/kg, ip.) and subsequent lethal (L: 10 mg/kg, ip.) doses of LPS alone or in combination with NB (100 mg/kg, ip.). Controls received saline (C) or NB. RESULTS: Preconditioning LPS conferred protection from a subsequent lethal LPS treatment. Importantly, the protective effect of LPS preconditioning was completely abolished by a concomitant treatment with NB. LPS induced a marked heat shock protein increase as demonstrated by Western blots of Hsp70 and Hsp90. NB alone also stimulated Hsp70 and Hsp90 mRNA but not protein expression. However, Hsp70 and Hsp90 protein induction in LPS-treated mice was abolished by a concomitant NB treatment, demonstrating a NB-induced impairment of the heat shock response to LPS preconditioning. CONCLUSION: LPS-induced heat shock protein induction and tolerance to a subsequent lethal LPS treatment was prevented by the Hsp90 inhibitor, novobiocin. Our findings demonstrate a critical role of Hsp90 in LPS signaling, and a potential involvement of the heat shock response in LPS-induced preconditioning

Loss of apical monocilia on collecting duct principal cells impairs ATP secretion across the apical cell surface and ATP-dependent and flow-induced calcium signals

Renal epithelial cells release ATP constitutively under basal conditions and release higher quantities of purine nucleotide in response to stimuli. ATP filtered at the glomerulus, secreted by epithelial cells along the nephron, and released serosally by macula densa cells for feedback signaling to afferent arterioles within the glomerulus has important physiological signaling roles within kidneys. In autosomal recessive polycystic kidney disease (ARPKD) mice and humans, collecting duct epithelial cells lack an apical central cilium or express dysfunctional proteins within that monocilium. Collecting duct principal cells derived from an Oak Ridge polycystic kidney (orpkTg737) mouse model of ARPKD lack a well-formed apical central cilium, thought to be a sensory organelle. We compared these cells grown as polarized cell monolayers on permeable supports to the same cells where the apical monocilium was genetically rescued with the wild-type Tg737 gene that encodes Polaris, a protein essential to cilia formation. Constitutive ATP release under basal conditions was low and not different in mutant versus rescued monolayers. However, genetically rescued principal cell monolayers released ATP three- to fivefold more robustly in response to ionomycin. Principal cell monolayers with fully formed apical monocilia responded three- to fivefold greater to hypotonicity than mutant monolayers lacking monocilia. In support of the idea that monocilia are sensory organelles, intentionally harsh pipetting of medium directly onto the center of the monolayer induced ATP release in genetically rescued monolayers that possessed apical monocilia. Mechanical stimulation was much less effective, however, on mutant orpk collecting duct principal cell monolayers that lacked apical central monocilia. Our data also show that an increase in cytosolic free Ca2+ primes the ATP pool that is released in response to mechanical stimuli. It also appears that hypotonic cell swelling and mechanical pipetting stimuli trigger release of a common ATP pool. Cilium-competent monolayers responded to flow with an increase in cell Ca2+ derived from both extracellular and intracellular stores. This flow-induced Ca2+ signal was less robust in cilium-deficient monolayers. Flow-induced Ca2+ signals in both preparations were attenuated by extracellular gadolinium and by extracellular apyrase, an ATPase/ADPase. Taken together, these data suggest that apical monocilia are sensory organelles and that their presence in the apical membrane facilitates the formation of a mature ATP secretion apparatus responsive to chemical, osmotic, and mechanical stimuli. The cilium and autocrine ATP signaling appear to work in concert to control cell Ca2+. Loss of a cilium-dedicated autocrine purinergic signaling system may be a critical underlying etiology for ARPKD and may lead to disinhibition and/or upregulation of multiple sodium (Na+) absorptive mechanisms and a resultant severe hypertensive phenotype in ARPKD and, possibly, other diseases

Pseudouridylation defect due to DKC1 and NOP10 mutations causes nephrotic syndrome with cataracts, hearing impairment, and enterocolitis

Research UK Innovation and Project; Cancer Research U

Angiotensin II activates plasminogen activator inhibitor-I promoter in renal tubular epithelial cells via the AT1 receptor

Background: Plasminogen activator inhibitor-1 (PAI-1) regulates normal extracellular matrix (ECM) metabolism and it is a key regulator of the fibrotic process. Both angiotensin II (Ang II) and angiotensin IV (Ang IV) have been reported to stimulate PAI-1 expression. It is not known how PAI-1 expression is regulated by the renin-angiotensin system (RAS) in renal tubular cells. Methods: To dissect signaling mechanisms contributing to the up-regulation of the PAI-1 promoter, porcine proximal tubular cells stably expressing the rabbit AT 1 receptor (LLC-PK/AT 1 ) were transiently transfected with a luciferase reporter construct containing the PAI-1 promoter. Promoter activation was assessed by measuring luciferase activity from cell lysates. Results: Ang II dose-dependently stimulated the transcriptional activity of the PAI-1 promoter in renal proximal tubular cells whereas Ang IV had no consistent effect on the promoter activity. Neither inhibition of the Extracellular Signal Regulated Kinase (ERK) cascade nor inhibition of the c-Jun-N- terminal Kinase (JNK) pathway did reduce the stimulation of the PAI-1 promoter by Ang II. However, genistein, a tyrosine kinase inhibitor blocked the effect of Ang II. Conclusion: Ang II but not Ang IV activates the PAI-1 promoter in renal proximal tubular cells and this effect is mediated by tyrosine kinases

Renin-angiotensin system affects endothelial morphology and permeability of renal afferent arteriole

The afferent arteriole (AA) is an important regulatory site of renal function and blood pressure. We have demonstrated endothelial fenestration and high permeability in the vicinity of renin granulated epithelioid cells in the juxtaglomerular portion of the afferent arteriole in different mammals. The permeability of fenestrated endothelium of afferent arteriole may be important in connection to various physiologic and pathophysiologic processes. We have assumed that the permeable fenestration may serve as a communication channel between the intravascular circulation and a pathway for renin secretion. Utilising the multiphoton image technique we were able to visualise the endothelial fenestration and renin granules of the in vitro microperfused AA and in vivo AA. We demonstrated that ferritin-positive, i.e., permeable portion of the afferent arteriole, under control conditions is on average 45 μm, which is about one-third to half of the total length of the afferent arteriole. The length of this portion is not constant and can change by physiologic and pharmacologic manipulation of renin formation. The permeability of the afferent arteriole is not changing only parallel with the pharmacologically stimulated renin secretion as already demonstrated in adult rats, but also with the change of renin appearance in afferent arteriole within the very first few days of life after birth. Independently from the age there is a significant correlation between the renin-positive and permeable portion of the AA. Further studies are necessary to clarify the physiological significance of afferent arteriolar permeability and its changes in the postnatal development of the kidney, as well as in correlation with activity of renin- angiotensin system

Data from: Determinants of between-year burrow re-occupation in a colony of the European Bee-eater Merops apiaster

Re-occupation of existing nesting burrows in the European bee-eater Merops apiaster has only rarely – and if so mostly anecdotically – been documented in the literature record, although such behavior would substantially save time and energy. In this study, we quantify burrow re-occupation in a German colony over a period of eleven years and identify ecological variables determining reuse probability. Of 179 recorded broods, 54% took place in a reused burrow and the overall probability that one of 75 individually recognized burrows would be reused in a given subsequent year was estimated as 26.4%. This indicates that between-year burrow reuse is a common behavior in the study colony which contrasts with findings from studies in other colonies. Furthermore, burrow re-occupation probability declined highly significantly with increasing age of the breeding wall. Statistical separation of within- and between-burrow effects of the age of the breeding wall revealed that a decline in re-occupation probability with individual burrow age was responsible for this and not a selective disappearance of burrows with high re-occupation probability over time. Limited duty cycles of individual burrows may be caused by accumulating detritus or decreasing stability with increasing burrow age. Alternatively, burrow fidelity may presuppose pair fidelity which may also explain the observed restricted burrow reuse duty cycles. A consequent next step would be to extend our within-colony approach to other colonies and compare the ecological circumstances under which bee-eaters reuse breeding burrows