Measurement of Revascularization in the Hind Limb After Experimental Ischemia in Mice.

Peripheral vascular disease is a major cause of morbidity and mortality, and is a consequence of impaired blood flow to the limbs. This arises due to the inability of the tissue to develop sufficiently functional collateral vessel circulation to overcome occluded arteries, or microvascular impairment. The mouse hind limb model of hind limb ischemia can be used to investigate the impact of different treatment modalities, behavioral changes, or genetic knockout. Here we described the model in detail, providing examples of adverse events, and details of ex vivo analysis of blood vessel density.

Emergency surgery for brainstem cavernoma haemorrhage with severe neurological presentation. Is it indicated and worthwhile?

Background: Brainstem Cavernoma (BSCM) haemorrhage is a complex condition, especially when patients present rapid neurological deterioration. Traditionally, these patients were initially treated by non-interventional means. Surgery was generally reserved for cases who presented a 'benign' evolution in a subacute/delayed fashion. Timing of surgery remains controversial. Since rebleeding is frequent and carries a high mortality, many of these patients do not tolerate this approach. Urgent/emergent surgery may be indicated and lifesaving.Methods: A single center experience is reported in which an aggressive approach was used with urgent/emergency surgery carried out on patients with BSCM haemorrhage and rapid neurological deterioration, ventilatory impairment and/or coma. A review of 5 consecutive cases where urgent/emergent surgery was performed is presented. The pre-operative status, pre- and post-operative examinations, surgical approach and neurological residual deficits/outcomes are reported.Results: Four females and one male with ages ranging from 36 to 66 years with rapid neurological deterioration, ventilatory impairment and/or coma were operated between 2011 and 2018. Favourable outcomes were observed with a modified Rankin Scale varying from 1 to 4. Cranial nerve deficits as well as motor and sensitive deficits were observed but all the patients recovered cognitive integrity.Conclusions: Our small series reveals an acceptable outcome with ultra-early surgery. This approach appears to be a valid option when there is rapid neurological deterioration, respiratory impairment and/or early onset coma. However, further studies are required to elucidate the optimal strategy.

Enhanced notch signaling modulates unproductive revascularization in response to nitric oxide-angiopoietin signaling in a mouse model of peripheral ischemia.

INTRODUCTION: Arteriolargenesis can be induced by concomitant stimulation of nitric Oxide (NO)-Angiopoietin receptor (Tie)-Vascular Endothelial Growth Factor (VEGF) signaling in the rat mesentery angiogenesis assay. We hypothesized that the same combination of exogenously added growth factors would also have a positive impact on arteriolargenesis and, consequently, the recovery of blood flow in a model of unilateral hindlimb ischemia. RESULTS AND METHODS: NO-Tie mice had faster blood flow recovery compared to control mice, as assessed by laser speckle imaging. There was no change in capillary density within the ischemic muscles, but arteriole density was higher in NO-Tie mice. Given the previously documented beneficial effect of VEGF signaling, we tested whether NO-Tie-VEGF mice would show further improvement. Surprisingly, these mice recovered no differently from control, arteriole density was similar and capillary density was lower. Dll4 is a driver of arterial specification, so we hypothesized that Notch1 expression would be involved in arteriolargenesis. There was a significant upregulation of Notch1 transcripts in NO-Tie-VEGF compared with NO-Tie mice. Using soluble Dll4 (sDll4), we stimulated Notch signaling in the ischemic muscles of mice. NO-Tie-sDll4 mice had significantly increased capillary and arteriole densities, but impaired blood flow recovery. CONCLUSION: These results suggest that Dll4 activation early on in revascularization can lead to unproductive angiogenesis and arteriolargenesis, despite increased vascular densities. These results suggest spatial and temporal balance of growth factors needs to be perfected for ideal functional and anatomical revascularisation.

Activation of Notch signaling by soluble Dll4 decreases vascular permeability via a cAMP/PKA-dependent pathway.

The Notch ligand delta-like ligand 4 (Dll4), upregulated by VEGF, is a key regulator of vessel morphogenesis and function, controlling tip and stalk cell selection during sprouting angiogenesis. Inhibition of Dll4 results in hypersprouting, nonfunctional, poorly perfused vessels, suggesting a role for Dll4 in the formation of mature, reactive, functional vessels, with low permeability and able to restrict fluid and solute exchange. We tested the hypothesis that Dll4 controls transvascular fluid exchange. A recombinant protein expressing only the extracellular portion of Dll4 [soluble Dll4 (sDll4)] induced Notch signaling in endothelial cells (ECs), resulting in increased expression of vascular-endothelial cadherin, but not the tight junctional protein zonula occludens 1, at intercellular junctions. sDll4 decreased the permeability of FITC-labeled albumin across EC monolayers, and this effect was abrogated by coculture with the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester. One of the known molecular effectors responsible for strengthening EC-EC contacts is PKA, so we tested the effect of modulation of PKA on the sDll4-mediated reduction of permeability. Inhibition of PKA reversed the sDll4-mediated reduction in permeability and reduced expression of the Notch target gene Hey1. Knockdown of PKA reduced sDLL4-mediated vascular-endothelial cadherin junctional expression. sDll4 also caused a significant decrease in the hydraulic conductivity of rat mesenteric microvessels in vivo. This reduction was abolished upon coperfusion with the PKA inhibitor H89 dihydrochloride. These results indicate that Dll4 signaling through Notch activation acts through a cAMP/PKA pathway upon intercellular adherens junctions, but not tight junctions, to regulate endothelial barrier function. NEW & NOTEWORTHY Notch signaling reduces vascular permeability through stimulation of cAMP-dependent protein kinase A.

Physiological Role of Vascular Endothelial Growth Factors as Homeostatic Regulators.

The vascular endothelial growth factor (VEGF) family of proteins are key regulators of physiological systems. Originally linked with endothelial function, they have since become understood to be principal regulators of multiple tissues, both through their actions on vascular cells, but also through direct actions on other tissue types, including epithelial cells, neurons, and the immune system. The complexity of the five members of the gene family in terms of their different splice isoforms, differential translation, and specific localizations have enabled tissues to use these potent signaling molecules to control how they function to maintain their environment. This homeostatic function of VEGFs has been less intensely studied than their involvement in disease processes, development, and reproduction, but they still play a substantial and significant role in healthy control of blood volume and pressure, interstitial volume and drainage, renal and lung function, immunity, and signal processing in the peripheral and central nervous system. The widespread expression of VEGFs in healthy adult tissues, and the disturbances seen when VEGF signaling is inhibited support this view of the proteins as endogenous regulators of normal physiological function. This review summarizes the evidence and recent breakthroughs in understanding of the physiology that is regulated by VEGF, with emphasis on the role they play in maintaining homeostasis. © 2017 American Physiological Society. Compr Physiol 8:955-979, 2018.

Differential regulation of blood flow-induced neovascularization and mural cell recruitment by vascular endothelial growth factor and angiopoietin signalling.

KEY POINTS: Combining nitric oxide (NO)-mediated increased blood flow with angiopoietin-1-Tie2 receptor signalling induces arteriolargenesis - the formation of arterioles from capillaries - in a model of physiological angiogenesis. This NO-Tie-mediated arteriolargenesis requires endogenous vascular endothelial growth factor (VEGF) signalling. Inhibition of VEGF signalling increases pericyte coverage in microvessels. Together these findings indicate that generation of functional neovasculature requires close titration of NO-Tie2 signalling and localized VEGF induction, suggesting that the use of exogenous VEGF expression as a therapeutic for neovascularization may not be successful. ABSTRACT: Signalling through vascular endothelial growth factor (VEGF) receptors and the tyrosine kinase with IgG and EGF domains-2 (Tie2) receptor by angiopoietins is required in combination with blood flow for the formation of a functional vascular network. We tested the hypothesis that VEGF and angiopoietin-1 (Ang1) contribute differentially to neovascularization induced by nitric oxide (NO)-mediated vasodilatation, by comparing the phenotype of new microvessels in the mesentery during induction of vascular remodelling by over-expression of endothelial nitric oxide synthase in the fat pad of the adult rat mesentery during inhibition of angiopoietin signalling with soluble Tie2 (sTie2) and VEGF signalling with soluble Fms-like tyrosine kinase receptor-1 (sFlt1). We found that NO-mediated angiogenesis was blocked by inhibition of VEGF with sFlt1 (from 881 ± 98% increase in functional vessel area to 279 ± 72%) and by inhibition of angiopoietin with sTie2 (to 337 ± 67%). Exogenous angiopoietin-1 was required to induce arteriolargenesis (8.6 ± 1.3% of vessels with recruitment of vascular smooth muscle cells; VSMCs) in the presence of enhanced flow. sTie2 and sFlt1 both inhibited VSMC recruitment (both 0%), and VEGF inhibition increased pericyte recruitment to newly formed vessels (from 27 ± 2 to 54 ± 3% pericyte ensheathment). We demonstrate that a fine balance of VEGF and angiopoietin signalling is required for the formation of a functional vascular network. Endogenous VEGF signalling prevents excess neovessel pericyte coverage, and is required for VSMC recruitment during increased nitric oxide-mediated vasodilatation and angiopoietin signalling (NO-Tie-mediated arteriogenesis). Therapeutic vascular remodelling paradigms may therefore require treatments that modulate blood flow to utilize endogenous VEGF, in combination with exogenous Ang1, for effective neovascularization.

Recurring flood distribution patterns related to short-term Holocene climatic variability.

Millennial- and multi-centennial scale climate variability during the Holocene has been well documented, but its impact on the distribution and timing of extreme river floods has yet to be established. Here we present a meta-analysis of more than 2000 radiometrically dated flood units to reconstruct centennial-scale Holocene flood episodes in Europe and North Africa. Our data analysis shows a general increase in flood frequency after 5000 cal. yr BP consistent with a weakening in zonal circulation over the second half of the Holocene, and with an increase in winter insolation. Multi-centennial length phases of flooding in UK and central Europe correspond with periods of minimum solar irradiance, with a clear trend of increasing flood frequency over the last 1000 years. Western Mediterranean regions show synchrony of flood episodes associated with negative phases of the North Atlantic Oscillation that are out-of-phase with those evident within the eastern Mediterranean. This long-term flood record reveals complex but geographically highly interconnected climate-flood relationships, and provides a new framework to understand likely future spatial changes of flood frequency.

Netrin-1 controls sympathetic arterial innervation.

Autonomic sympathetic nerves innervate peripheral resistance arteries, thereby regulating vascular tone and controlling blood supply to organs. Despite the fundamental importance of blood flow control, how sympathetic arterial innervation develops remains largely unknown. Here, we identified the axon guidance cue netrin-1 as an essential factor required for development of arterial innervation in mice. Netrin-1 was produced by arterial smooth muscle cells (SMCs) at the onset of innervation, and arterial innervation required the interaction of netrin-1 with its receptor, deleted in colorectal cancer (DCC), on sympathetic growth cones. Function-blocking approaches, including cell type-specific deletion of the genes encoding Ntn1 in SMCs and Dcc in sympathetic neurons, led to severe and selective reduction of sympathetic innervation and to defective vasoconstriction in resistance arteries. These findings indicate that netrin-1 and DCC are critical for the control of arterial innervation and blood flow regulation in peripheral organs.

Temporal changes in microvessel leakiness during wound healing discriminated by in vivo fluorescence recovery after photobleaching.

Regeneration of injured tissue is a dynamic process, critically dependent on the formation of new blood vessels and restructuring of the nascent plexus. Endothelial barrier function, a functional correlate of vascular restructuring and maturation, was quantified via intravital microscopic analysis of 150 kDa FITC-dextran-perfused blood vessels within discrete wounds created in the panniculus carnosus (PC) muscle of dorsal skinfold chamber (DSC) preparations in mice. Time to recovery of half-peak fluorescence intensity (t(1/2)) within individual vessel segments in three functional regions of the wound (pre-existing vessels, angiogenic plexus and blind-ended vessels (BEVs)) was quantified using in vivo fluorescence recovery after photobleaching (FRAP) and linear regression analysis of recovery profiles. Plasma flux across the walls of new vessel segments, particularly BEVs, was greater than that of pre-existing vessels at days 5-7 after injury (P < 0.05). TNP-470 reduced the permeability of BEVs at the leading edge of the advancing vascular plexus as measured by the decrease in luminal t(1/2) (P < 0.05), confirming the utility of FRAP as a quantitative measure of endothelial barrier function. Furthermore, these data are suggestive of a role for TNP-470 in selection for less leaky vascular segments within healing wounds. Increased FITC-dextran leakage was observed from pre-existing vessels after treatment with TNP-470 (P < 0.05), consistent with induction of transient vascular damage, although the significance of this finding is unclear. Using in vivo FRAP this study demonstrates the relationship between temporal changes in microvascular macromolecular flux and the morphology of maturing vascular segments. This combination of techniques may be useful to assess the therapeutic potential of angiogenic agents in restoring pre-injury levels of endothelial barrier function, following the establishment of a functional vascular plexus such as in models of wounding or tumour development.

Dynamics of angiogenesis during wound healing: a coupled in vivo and in silico study.

OBJECTIVE: The most critical determinant of restoration of tissue structure during wound healing is the re-establishment of a functional vasculature, which largely occurs via angiogenesis, specifically endothelial sprouting from the pre-existing vasculature. MATERIALS AND METHODS: We used confocal microscopy to capture sequential images of perfused vascular segments within the injured panniculus carnosus muscle in the mouse dorsal skin-fold window chamber to quantify a range of microcirculatory parameters during the first nine days of healing. This data was used to inform a mathematical model of sequential growth of the vascular plexus. The modeling framework mirrored the experimental circular wound domain and incorporated capillary sprouting and endothelial cell (EC) sensing of vascular endothelial growth factor gradients. RESULTS: Wound areas, vessel densities and vessel junction densities obtained from the corresponding virtual wound were in excellent agreement both temporally and spatially with data measured during the in vivo healing process. Moreover, by perturbing the proliferative ability of ECs in the mathematical model, this leads to a severe reduction in vascular growth and poor healing. Quantitative measures from this second set of simulations were found to correlate extremely well with experimental data obtained from animals treated with an agent that targets endothelial proliferation (TNP-470). CONCLUSION: Our direct combination and comparison of in vivo longitudinal analysis (over time in the same animal) and mathematical modeling employed in this study establishes a useful new paradigm. The virtual wound created in this study can be used to investigate a wide range of experimental hypotheses associated with wound healing, including disorders characterized by aberrant angiogenesis (e.g., diabetic models) and the effects of vascular enhancing/disrupting agents or therapeutic interventions such as hyperbaric oxygen.

Experimental and theoretical modelling of blind-ended vessels within a developing angiogenic plexus.

Angiogenic sprouts at the leading edge of an expanding vascular plexus are recognised as major regulators of the structure of the developing network. Early in sprout development, a vascular lumen is often evident which communicates with the parent vessel while the distal tip is blind-ended. Here we describe the temporal evolution of blind-ended vessels (BEVs) in a small wound made in the panniculus carnosus muscle of a mouse viewed in a dorsal skin-fold window-chamber model with intra-vital microscopy during the most active period of angiogenesis (days 5-8 after injury). Although these structures have been mentioned anecdotally in previous studies, we observed BEVs to be frequent, albeit transient, features of plexus formation. Plasma leakage into the surrounding extracellular matrix occurring from these immature conduits could play an important role in preparing hypoxic tissue for vascular invasion. Although sprout growth is likely to be regulated by its flow environment, the parameters regulating flow into and through BEVs have not been characterised in situ. Longitudinal data from individual animals show that the number of BEVs filled with plasma alone peaks at day 7, when they can exceed 150 microm in length. Additionally, BEVs greater than 40 microm in length are more likely to be filled with stationary erythrocytes than with plasma alone. Using a mathematical model, we show how the flux of 150 kD fluorinated (FITC-) dextran through an individual plasma-filled BEV is related to its geometry being determined primarily by its surface area; by fitting theoretical intensity values to experimental data we assess the permeability of the vessel to FITC-dextran. Plasma skimming provides a mechanistic explanation for the observation that BEVs with larger surface area are more likely to recruit erythrocytes.

Effects of antenatal betamethasone on human fetal branch pulmonary artery flow velocity waveforms.

OBJECTIVE: To evaluate the effect of antenatal betamethasone on fetal branch pulmonary artery flow velocity waveforms. METHODS: Betamethasone 12 mg i.m. in 2 injections 24 h apart was given to 25 pregnant women at risk of preterm delivery. Pulsatility (PI) and resistance (RI) indexes were measured at the proximal, middle and distal segments of the fetal pulmonary artery before and repeatedly 1, 2 and 7 days after the first dose. ANOVA for repeated measures and the general linear model were used to statistically analyze the results. RESULTS: Blood flow resistance decreased after betamethasone treatment at both the middle (p = 0.001 and p = 0.02 for RI and PI, respectively) and the proximal (p = 0.03 for RI) segments returning to basal values after 7 days. CONCLUSIONS: Antenatal betamethasone decreases fetal branch pulmonary artery vascular resistance.

Polimorfismo genético del pepsinógeno A urinario en la provincia de Villa Clara (Cuba) / Genetic polyorphism of urinary pepsinogen A in Villa Clara Province (Cuba)

Los patrones electroforéticos del pepsinógeno A (PGA) se determinaron en una muestra de 401 orinas de niños sanos de la raza blanca. En nuestro estudio sólo se consideró la presencia o ausencia del fenotipo electroforético 5 (Pg5) y se señaló como Pg5+ o como Pg5, respectivamente. Dicha isoproteína está determinada por el gen D, la cual se comporta como un carácter autosómico dominante. Las frecuencias fenotípicas encontradas fueron: Pg5+ = ,850 y Pg5 = ,150. La frecuencia génica calculada para el alelo D es de ,613. Debido a la asociación entre el cáncer del estómago y el fenotipo Pg5+, el mismo pudiera definir un grupo de riesgo en nuestra población

Polimorfismo genético del pepsinógeno A urinario en la provincia de Villa Clara (Cuba) / Genetic polymorphism of urinary pepsinogen A in Villa Clara Province (Cuba)

Los patrones electroforéticos del pepsinógeno A (PGA) se determinaron en una muestra de 401 orinas de niños sanos de la raza blanca. En nuestro estudio sólo se consideró la presencia o ausencia del fenotipo electroforético 5 (Pg5) y se señaló como Pg5+ o como Pg5, respectivamente. Dicha isoproteína está determinada por el gen D, la cual se comporta como un carácter autosómico dominante. Las frecuencias fenotípicas encontradas fueron: Pg5+ = ,850 y Pg5 = ,150. La frecuencia génica calculada para el alelo D es de ,613. Debido a la asociación entre el cáncer del estómago y el fenotipo Pg5+, el mismo pudiera definir un grupo de riesgo en nuestra población