Shear stress-induced angiogenesis in mouse muscle is independent of the vasodilator mechanism and quickly reversible.

Aim: Is modulation of skeletal muscle capillary supply by altering blood flow due to a presumptive shear stress response per se, or dependent on the vasodilator mechanism? Methods: The response to four different vasodilators, and cotreatment with blockers of NO and prostaglandin synthesis, was compared. Femoral artery blood flow was correlated with capillary-to-fibre ratio (C:F) and protein levels of putative angiogenic compounds. Results: All vasodilators induced a similar increase in blood flow after 14 days, with a similar effect on C:F (1.62 ± 0.05, 1.60 ± 0.01, 1.57 ± 0.06, 1.57 ± 0.07, respectively, all P < 0.05 vs. control 1.20 ± 0.01). Concomitant inhibitors revealed differential effects on blood flow and angiogenesis, demonstrating that a similar response may have different signalling origins. The time course of this response with the most commonly used vasodilator, prazosin, showed that blood flow increased from 0.40 mL min−1 to 0.61 mL min−1 by 28 days (P < 0.05), dropped within 1 week after the cessation of treatment (0.54 mL min−1; P < 0.05) and returned to control levels by 6 weeks. In parallel with FBF, capillary rarefaction began within 1 week (P < 0.05), giving C:F values similar to control by 2 weeks. Of the dominant signalling pathways, prazosin decreased muscle VEGF, but increased its cognate receptor Flk-1 (both P < 0.01); levels of eNOS varied with blood flow (P < 0.05), and Ang-1 initially increased, while its receptor Tie-2 was unchanged, with only modest changes in the antiangiogenic factor TSP-1. Conclusion: Hyperaemia-induced angiogenesis, likely in response to elevated shear stress, is independent of the vasodilator involved, with a rapid induction and quick regression following the stimulus withdrawal

On the functional overlap between complement and anti-microbial peptides

Intriguingly, activated complement and anti-microbial peptides share certain functionalities; lytic, phagocytic, and chemo-attractant activities and each may, in addition, exert cell instructive roles. Each has been shown to have distinct LPS detoxifying activity and may play a role in the development of endotoxin tolerance. In search of the origin of complement, a functional homolog of complement C₃ involved in opsonization has been identified in horseshoe crabs. Horseshoe crabs possess anti-microbial peptides able to bind to acyl chains or phosphate groups/saccharides of endotoxin, LPS. Complement activity as a whole is detectable in marine invertebrates. These are also a source of anti-microbial peptides with potential pharmaceutical applicability. Investigating the locality for the production of complement pathway proteins and their role in modulating cellular immune responses are emerging fields. The significance of local synthesis of complement components is becoming clearer from in vivo studies of parenchymatous disease involving specifically generated, complement-deficient mouse lines. Complement C₃ is a central component of complement activation. Its provision by cells of the myeloid lineage varies. Their effector functions in turn are increased in the presence of anti-microbial peptides. This may point to a potentiating range of activities, which should serve the maintenance of health but may also cause disease. Because of the therapeutic implications, this review will consider closely studies dealing with complement activation and anti-microbial peptide activity in acute inflammation (e.g., dialysis-related peritonitis, appendicitis, and ischemia)

A new mechanism for the regulation of endothelial functions.

1. Titel-Inhaltsverzeichnis................................................ 1 2. Einleitung..................................................................... 7 3. Material und Methoden............................................. 36 4. Ergebnisse.................................................................. 63 5. Diskussion.................................................................. 86 6. Zusammenfassung-Summary................................... 111 7. Literaturverzeichnis................................................. 114 8. Abkürzungsverzeichnis............................................ 132 9. Anhang..................................................................... 134Die strömungsregulierte Genexpression im Endothel kann eine entscheidende Rolle für die Steuerung angioadaptiver Prozesse (z.B. Angiogenese) spielen. Einige Mitglieder der Thrombospondin (TSP) type 1 repeats (TSR) Familie weisen eine starke anti-angiogene Wirkung auf, die zum Teil über die Bindung der TSR Domäne an den CD36 Rezeptor vermittelt wird. In dieser Arbeit wurde die strömungsregulierte endotheliale Expression verschiedener TSR Proteine und des CD36 Rezeptors untersucht. Humane Endothelzellen (HUVEC, HCMEC) wurden mit einem Kegel-Platte-System unter verschiedenen Strömungsbedingungen kultiviert. Die Expression ausgewählter TSR Mitglieder (TSP-1/ -2, METH-1/ -2, �brain specific angiogenic inhibitor�-1, Prokollagen I N-Proteinase, Properdin) und des CD36 Rezeptors wurde mittels RT-PCR, Real-time PCR, Northern Blot und Western Blot untersucht. Die Expression von TSP-1, METH-1 und CD36 wurde darüber hinaus in einem in vivo Modell der strömungsinduzierten Angiogenese (Prazosin Modell; Maus) mittels Genearrays und Western Blot analysiert. Von den untersuchten angiogeneserelevanten Gene wurden sowohl in vitro als in vivo TSP-1, METH-1 und der CD36 Rezeptor durch Strömungsveränderungen moduliert. Die Expression von TSP-1 und CD36 wird durch Strömungsstillstand hochreguliert. Die durch Bindung von TSP-1 an CD36 induzierte endotheliale Apoptose, könnte daher zum Abbau nicht-perfundierter Blutgefäße beitragen. Die Expression von METH-1 wurde durch Strömung induziert. METH-1 kann die Angiogenese und die Proliferation von Endothelzellen hemmen. Unter diesem Aspekt könnte METH-1 zur Stabilisierung gut perfundierter Gefäße beitragen. Bei komplexeren angioadaptiven Vorgängen, wie der VEGF-induzierten Kapillar- sprossung, lässt sich eine mögliche zeitversetzte Wirkung von TSP-1/CD36 und METH-1 postulieren. In der ersten Phase, bei turbulenter Strömung/ Strömungsstillstand und in Anwesenheit von VEGF, wäre die Suppression von METH-1 für Sprossung und endotheliale Proliferation entscheidend. Bei Anschluss der Sprosse an ein weiteres Gefäß würde METH-1 durch die einsetzende Strömung induziert und das Gefäß stabilisiert. Bei anhaltendem Strömungsstillstand in einer blind endenden Sprosse würde dagegen TSP-1/CD36 induziert werden und durch endotheliale Apoptose der Gefäßabbau unterstützt. Die Expression von Properdin, einem Mitglied der TSR Familie, das die Angiogenese nicht moduliert, wird durch Strömung in Endothelzellen induziert. Damit ist das Endothel die bisher einzige bekannte Quelle dieses Regulators der Komplementaktivierung im Plasma. Die erhöhte Expression von Properdin durch turbulente Strömung weist auf seine mögliche Mitwirkung bei Entstehung und Fortschreiten der Arteriosklerose hin. [if !supportEmptyParas] [endif]The flow-dependent modulation of endothelial gene expression should play a key role for the regulation of angioadaptive processes (i.e. angiogenesis) in response to altered hemodynamic conditions. Several members of the Thrombospondin (TSP) type-1 repeat (TSR) family are able to inhibit angiogenesis, especially through the binding to the CD36 receptor. Aim of this study was the analysis of the flow-dependent modulation of TSR genes and of the CD36 receptor. Human endothelial cells (HUVEC, HCMEC) were exposed in vitro to different flow conditions using a cone-and-plate system. The expression of TSP-1/ -2, METH-1/ -2, brain specific angiogenic inhibitor-1, procollagen I N-proteinase, Properdin and CD36 was analysed by RT-PCR, Real-time PCR, Northern blot and Western blot. The expression of TSP-1, METH-1 and CD36 was further analysed in an in vivo model of flow induced angiogenesis (Prazosin Model) using gene arrays and Western blotting. Among the analysed angiogenesis-relevant genes, the expression of TSP-1, METH-1 and CD36 was modulated in response to altered flow conditions both in vitro and in vivo. TSP-1 and CD36 were induced in absence of flow. Since the binding of TSP-1 to the CD36 receptor can induce endothelial apoptosis, their increased expression in not perfused vessels (i.e. by stenosis) could contribute to vessel degradation. The expression of METH-1 was induced by flow. Considering the anti-angiogenic and anti-proliferative features of METH-1, its expression in presence of flow may represent a mechanism contributing to the stabilization of well perfused vessels. By more complex angioadaptive processes, like the VEGF-induced sprouting, the effects of TSP-1/CD36 and METH-1 could be temporarily differentiated. In the first phase, by turbulent/no flow conditions and in presence of VEGF, the expression of TSP-1/CD36 remains low and the inhibition of METH-1 would permit sprouting and endothelial cell proliferation. After connection of the sprout to a further vessel the expression of METH-1 would be up-regulated by flow and the new vessel stabilized. In absence of this connection, the induction of TSP-1/CD36 by prolonged absence of flow would contribute to the degradation of the blind-ending sprout. The expression of properdin, a member of the TSR family which does not affect angiogenesis, is modulated by flow exposure. The induction of this complement activating factor proposes the endothelium as the only so far known source for plasma properdin. The increased synthesis of properdin by turbulent flow conditions suggests its involvement in the initiation/progression of arteriosclerosis

Hypertension, aging, and myocardial synthesis of heat-shock protein 72

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 different 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% 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

Heat shock protein changes in hibernation: A similarity with heart failure?

Myocardial hibernation is an adaptive phenomenon occurring during ischaemia. Patients with hibernating myocardium often have a history of an acute ischaemic insult, followed by prolonged hypoperfusion and symptoms of congestive heart failure (CHF), which is a complex syndrome involving several adaptational mechanisms. We tested the hypothesis that these two conditions evoke the myocardial expression of heat shock protein 72 (hsp72) as an adaptive response at the molecular level. Short-term acute hibernation was induced in isolated and perfused rat hearts subjected to 8 min total ischaemia followed by 292 min low-flow ischaemia (coronary now: 1.0 ml/min), followed by 60 min of reperfusion. Total ischaemia caused quiescience. Subsequent low-now resulted in a temporal early increase of lactate release, no re-establishment of developed pressure, no increase in diastolic pressure. Reperfusion resulted in 85.7 ± 7.2% recovery of developed pressure, a small washout of lactate and CPK, no contracture, confirming that viability was maintained despite prolonged hypoperfusion. This sequence of events was linked to an increase in hsp72 content in the right (from 18.1 ± 3.8% to 34.6 ± 2.3%, P &lt; 0.01) and left (from 19.7 ± 2.6% to 37.6 ± 3.3%, P &lt; 0.01) ventricles. Three-hundred min of low-now perfusion of the rat heart in absence of the short period of total ischaemia caused irreversible damage and failed to induced hsp72. CHF was induced in rats by intraperitoneal administration of monocrotaline. As a result, right ventricular weight increased from 171.3 ± 7.2 to 412.3 ± 18.7 mg, P &lt; 0.001, peripheral and pleural effusion were evident and measurable, plasma arterial natriuretic peptide increased from 15.2 ± 1.9 to 123.5 ± 5.4 pg/ml, P &lt; 0.001, confirming the occurrence of the syndrome of CHF. This was concomitant with significant expression of hsp72, more evident in the right (from 5.0 ± 0.9% to 39.4 ± 1.6%, P &lt; 0.001) than in the left (from 3.5 ± 0.6% to 13.0 ± 1.2%, P &lt; 0.001) ventricle. These data suggest that an adaptational process occurs at myocardial level during either hibernation or CHF. The expression of hsp72 could be viewed as a stereotyped adaptational reaction of the cardiac cell to stress conditions

Shear stress modulates the expression of thrombospondin-1 and CD36 in endothelial cells in vitro and during shear stress-induced angiogenesis in vivo

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