ADAMTS1 inhibits capillary sprouting, and since capillary sprouts do not experience the shear stress caused by blood flow, this study undertook to clarify the relationship between shear stress and ADAMTS1. It was found that endothelial cells exposed to shear stress displayed a strong upregulation of ADAMTS1, dependent upon both the magnitude and duration of their exposure. Investigation of the underlying pathways demonstrated involvement of phospholipase C, phosphoinositide 3‐kinase, and nitric oxide. Forkhead box protein O1 was identified as a likely inhibitor of the system, as its knockdown was followed by a slight increase in ADAMTS1 expression. In silico prediction displayed a transcriptional binding site for Forkhead box protein O1 in the promotor region of the ADAMTS1 gene, as well as sites for nuclear factor 1, SP1, and AP‐1. The anti‐angiogenic effects of ADAMTS1 were attributed to its cleavage of thrombospondin 1 into a 70‐kDa fragment, and a significant enhancement of this fragment was indeed demonstrated by immunoblotting shear stress‐treated cells. Accordingly, scratch wound closure displayed a slowdown in conditioned medium from shear stress‐treated endothelial cells, an effect that could be completely blocked by a knockdown of thrombospondin 1 and partially blocked by a knockdown of ADAMTS1. Non‐perfused capillary sprouts in rat mesenteries stained negative for ADAMTS1, while vessels in the microcirculation that had already experienced blood flow yielded the opposite results. The shear stress‐dependent expression of ADAMTS1 in vitro was therefore also demonstrated in vivo and thereby confirmed as a mechanism connecting blood flow with the regulation of angiogenesis. J. Cell. Physiol. 226: 350–361, 2011. © 2010 Wiley‐Liss, Inc.

During and after prolonged bed rest, changes in bone metabolic markers occur within 3 days. Resistive vibration exercise during bed rest impedes bone loss and restricts increases in bone resorption markers whilst increasing bone formation.

While vascular endothelial cells are normally exposed to shear stress, there is no flow and consequently reduced shear stress in capillary sprouts. To explore, if this lack in mechano‐stimulation alters the endothelial phenotype, we analysed the influence of shear stress on the activity of transcription factor FOXO1 and the expression of its target gene angiopoietin‐2 (ang2), an antagonist of the endothelial tyrosine kinase receptor tie2.Endothelial cells were exposed to shear stress using a cone‐and‐plate system with or without inhibition of PI3K (using LY294,002) or FOXO1 (using siRNA) and analysed by real time RT‐PCR, Western blots, immunoprecipitation, and fluorescence microscopy.Shear stress (6°dyn/cm2) increased phosphorylation of Akt and FOXO1 in a PI3K‐dependent manner and FOXO1 was translocated out of the nucleus. In addition, expression of FOXO1 on the mRNA‐ and protein level was strongly reduced by shear stress. Suppression of FOXO1 by shear stress was accompanied by a decrease of ang2‐mRNA and ‐protein, which was similarly be achieved by siRNA against FOXO1. Following flow stop, ang2‐mRNA expression increased within two hours to steady state values under static conditions.So, in capillary sprouts, lacking shear stress activates endothelial FOXO1 by which ang2 is rapidly induced. Thus, the tie2‐system will be inactivated during capillary sprouting, which may turn the vessel wall more susceptible to additional angiogenic factors. By contrast, the onset of flow will activate the tie2‐system and thereby contribute to stabilize the vessel wall.

Transcription factor Foxo-1 can be inactivated via Akt-mediated phosphorylation. Since shear stress activates Akt, we determined whether Foxo-1 and the Foxo-1-dependent, angiogenesis-related Ang-2/Tie2-system are influenced by shear stress in endothelial cells. Expression of Foxo-1 and its target genes p27Kip1 and Ang-2 was decreased under shear stress (6dyn/cm(2), 24h), nuclear exclusion of Foxo-1 by phosphorylation increased. eNOS and Tie2 were upregulated. No effects on Ang-1 expression were detected. In conclusion, Foxo-1 and Ang-2/Tie2 are part of the molecular response to shear stress, which may regulate angiogenesis.

Capillary sprouting angiogenesis seems to be inhibited in well perfused microvessels. This study was done to explore the role of METH-1 in endothelial cells under these conditions. Human umbilical vein EC (HUVEC) were exposed to different flow conditions using a cone-and-plate apparatus while oxygen tension was varied between 20 mmHg and 150 mmHg. Gene expression was analyzed by RT-PCR, Northern, and Western blot. Conditioned media from flow-exposed or control HUVEC were tested by a scratch wound assay. In whole mounts of the rat mesentery, the expression of ADAMTS-1 was analysed by immunohistochemistry. Exposure of EC to shear stress (0.6–6 dyn·cm−2) results in a force-dependent and long lasting (up to 72 h) substantial up-regulation of METH-1 expression and secretion. This effect is fully reversible by restoration of no-flow conditions. A threshold shear stress of 1.0–2.0 dyn·cm−2 (24 h) is required for a significant up-regulation. At 100 mmHg oxygen tension and 6 dyn·cm−2 the expression of METH-1 is about 40-fold up-regulated compared to 20 mmHg oxygen tension and no flow. Closure of scratch wounds is significantly slowed down by conditioned medium from flow exposed cells. As predicted from these in vitro data, strong expression of ADAMTS-1 is observed in all segments of microvascular networks of the rat mesentery (wall shear stress 10–100 dyn·cm−2, oxygen tension 60–90 mmHg). In conclusion, there is strong inhibition of sprouting angiogenesis in the microcirculation by METH-1 under normal conditions. This inhibition may be suspended during reduced perfusion especially in combination with low oxygen tension. The expression characteristics of METH-1 indicate, that it may have a crucial role in matching angiogenesis to tissue needs. Supp. by DFG ZA 184/3-2

Binding of thrombospondin-1 (TSP-1) to the CD36 receptor inhibits angiogenesis and induces apoptosis in endothelial cells (EC). Conversely, matrix-bound TSP-1 supports vessel formation. In this study we analyzed the shear stress-dependent expression of TSP-1 and CD36 in endothelial cells in vitro and in vivo to reveal its putative role in the blood flow-induced remodelling of vascular networks. Shear stress was applied to EC using a cone-and-plate apparatus and gene expression was analyzed by RT-PCR, Northern and Western blot. Angiogenesis in skeletal muscles of prazosin-fed (50 mg/l drinking water; 4 d) mice was assessed by measuring capillary-to-fiber (C/F) ratios. Protein expression in whole muscle homogenates (WMH) or BS-1 lectin-enriched EC fractions (ECF) was analyzed by Western blot. Shear stress downregulated TSP-1 and CD36 expression in vitro in a force- and time-dependent manner sustained for at least 72 h and reversible by restoration of no-flow conditions. In vivo, shear stress-driven increase of C/F in prazosin-fed mice was associated with reduced expression of TSP-1 and CD36 in ECF, while TSP-1 expression in WMH was increased. Down-regulation of endothelial TSP-1/CD36 by shear stress suggests a mechanism for inhibition of apoptosis in perfused vessels and pruning in the absence of flow. The increase of extra-endothelial (e.g. matrix-bound) TSP-1 could support a splitting type of vessel growth.

We determined the temperature-induced synthesis of the 72-kD heat-shock protein (hsp72) in hearts of normotensive and spontaneously hypertensive rats (SHR) subjected to whole-body hyperthermia (42.0±0.5°C for 15 minutes). The animals were studied at three diiferent ages: young (2 months), adult (6 months), and old (18 months). The hsp72 was determined by Western blot analysis using a monoclonal antibody. The results were calculated densitometrically as a percentage of a commercial standard. Young SHR responded to hyperthermic stress with increased synthesis of hsp72 compared with age-matched normotensive rats (298.8 ±70.0% versus 88.3±25.5%). This trend was maintained in adult rats (118.1±31.0% A common feature of genetically hypertensive animals is a greater sensitivity to environmental temperature with respect to that of their normotensive control counterparts.The increased thermosensitivity is genetically linked with hypertension and at the molecular level is characterized by an overexpression of heat-shock proteins, a group of highly conserved proteins synthesized during stress and involved in cellular protection. Increased synthesis of the 72-kD heat-shock protein (hsp72) is a specific marker of the stress response in the heart as hsp72 is induced in the myocardium after heat treatment, ischemia, hemodynamic overload, or hypoxj a i3-i6 T n e synthesis of hsp72 in the myocardium of heat-shocked hypertensive animals has never been fully studied. This is particularly relevant because recently the induction of hsp72 synthesis has been shown to protect the myocardium against ischemia," a pathological condition often associated with hypertension. During aging, the heart undergoes progressive functional alterations and becomes more vulnerable to ischemic insult. This is true of the hypertrophic heart of the spontaneously hypertensive rat (SHR).Several studies show an age-dependent decrease in the expression of heat-shock protein in response to environmental stress in both isolated cell systemsand tissues.The first aim of this study was to determine the synthesis of hsp72 in the heart of normotensive WistarKyoto (WKY) rats and SHR subjected to whole-body hyperthermia. The second aim was to investigate the effects of aging on the synthesis of hsp72. Received April 26, 1994; accepted in revised form July 26, 1994. From Cattedra di Cardiologia, Universita' di Brescia (R.F.), and Fondazione Clinica del Lavoro, Centro di Fisiopatologia Cardiovascolare Salvatore Maugeri, Gussago, Brescia, Italy. Reprint requests to Roberto Ferrari, Cattedra di Cardiologia, Universita' degli Studi, c/o Spedali Civili, P. le Spedali Civili, 1, 25123 Brescia, Italy. © 1994 American Heart Association, Inc. versus 54.8±21.3%) but not in old rats (65.3±29.4% versus 43.6±15.1%). Aging caused a reduction of hsp72 expression in response to hyperthermic stress in both SHR (4.6-fold) and normotensive rats (twofold). These data show that hearts of young and adult SHR respond to heat shock with enhanced synthesis of hsp72. This abnormal response, attenuated by aging, is independent of the presence and degree of hypertension or hypertrophy and is potentially linked to the genetic determination of the disease. (Hypertension. 1994^4:620-624.)

Properdin is a positive regulator of the alternative pathway of complement activation. It can be released by peripheral blood cells but is not synthesized in the liver and the physiological source of properdin in plasma is unknown. The endothelium is an extra-hepatic source for several complement components and shear stress can modulate their expression. The aim of this study was to analyze shear stress-exposed endothelial cells (EC) as physiological source for plasma properdin. Human umbilical vein EC (HUVEC) and human cardiac microvascular EC (HCMEC) were exposed to shear stress using a cone-and-plate apparatus and properdin expression was analyzed by RT-PCR, Northern, and Western blot. mRNA for properdin is barely detectable in untreated EC but strongly induced by laminar shear stress exposure (6 dyn/cm(2); 24 h). Properdin is induced also at the protein level and is released in the extracellular compartment. Properdin up-regulation requires a shear stress of 2-3 dyn/cm(2), is not transient, and is reversible by restoration of static conditions. Turbulent flow exposure results in two times higher induction of properdin than laminar flow exposure. The ability of endothelial cells exposed to shear stress to synthesize properdin proposes the endothelium as physiological source for plasma properdin and suggests a link between flow conditions and the modulation of the alternative pathway. Furthermore, the stronger properdin induction by turbulent flow may suggest an involvement in the pathology of atherosclerosis.