Increase In Vascular Area Research Articles

Abstract 17144: Pi3 Kinase Activation by Platelet-Derived Purified Exosome Product to Induce Angiogenesis

Introduction: Platelets are heavily implicated in angiogenesis; however, the underlying mechanism remains relatively uncharacterized. The goal of this study was to evaluate the angiogenic potency of a platelet-derived regenerative exosome product (PEP) in an effort to develop a novel treatment for peripheral arterial disease. Here, we evaluated the mechanistic basis of PEP in angiogenesis and it’s use In Vivo in a rodent hind limb ischemia model. Methods/Results: Under Current Good Manufacturing Practices, conditioned medium from apheresis platelet cultures was purified to acquire the exosomes. PEP, enriched in exosome markers CD63, CD9 and Flotillin, induced human umbilical vein endothelial cell (HUVEC) proliferation, migration, and vascular tube formation In Vitro. Proteomic evaluation of PEP highlighted receptor tyrosine kinase (RTK) pathways and western blot validation demonstrated induction of phospho-MAPK and phospho-AKT following PEP treatment of HUVECs. Systematic evaluation of RTK receptor involvement was performed using small molecule inhibitors and receptor knock-down in HUVECs, ruling out RTK activation as a mode of action. However, small molecule inhibition of PI3 kinase demonstrated elimination of PEP-induced AKT phosphorylation and reduction in PEP-induced angiogenesis In Vitro measured by HUVEC proliferation and migration. In Vivo, evaluation of angiogenesis was performed in a rodent model of hindlimb ischemia treated with PEP embedded in fibrin glue (PEP/TISSEEL). Blood flow was evaluated by SPY angiography on day 0 pre- and post-operation, day 21, and day 28. Animals treated with PEP/TISSEEL showed a significant increase in blood flow compared to sham and TISSEEL treated animals (percent perfusion at 28 days: sham 40.9%, TISSEEL 44.3%, PEP/TISSEEL 100.2%), with significant increase in vascular area on histology (at 28 days vascular area (μm 2 )/muscle area(μm 2 ): healthy control 12.71, sham 4.66, TISSEEL 4.76, PEP/TISSEEL 15.54). Conclusions: Exosome driven activation of PI3 kinase can induce angiogenesis both In Vitro and In Vivo. PEP provides a novel, clinical-grade, off-the-shelf therapy to promote angiogenesis for the treatment of peripheral arterial disease.

Histological Analysis of Regenerative Properties in Human Glenoid Labral Regions

Background: The healing capacity of the human glenoid labrum varies by tear location. Current evidence suggests that the healing capacity of meniscal and cartilage injuries relates to cellular composition and vascularity. However, little is known about the histological characteristics of the glenoid labrum and how they may affect healing potential in specific anatomic regions. Hypothesis: Regenerative characteristics of the glenoid labrum differ based on the anatomic region. Study Design: Descriptive laboratory study. Methods: Human glenoid labra from fresh unpreserved cadavers were transversely sectioned in different anatomic regions. Masson trichrome stain was used to determine dense and loose extracellular matrix regions and vessel densities. Hematoxylin and eosin, Ki-67+, and CD90+/CD105+ stains were performed to determine total, proliferative, and progenitor cell densities, respectively. Regression models demonstrated relationships between vascular area, progenitor cell quantity, and probability of successful operation. Results: Among all labral aspects, the superior glenoid labrum had the highest percentage (56.8% ± 6.9%) of dense extracellular matrix or avascular tissue (P < .1). The vascular region of the superior labrum had the fewest total cells (321 ± 135 cells/mm2; P < .01) and progenitor cells (20 ± 4 cells/mm2; P < .001). Vascular area was directly correlated with progenitor cell quantity (P = .006002). An increase in probability of successful operation was associated with a linear increase in vascular area (R2 = 0.765) and an exponential increase in progenitor cell quantity (R2 = 0.795). Subsequently, quadratic models of vascularity and progenitor cell quantity around the labral clock were used to assess relative healing potential. Quadratic models for percentage vascular area (P = 6.35e-07) and weighted progenitor cell density (P = 3.03e-05) around the labral clock showed that percentage vascular area and progenitor cell quantity increased as labral tissue neared the inferior aspect and diminished near the superior aspect. Conclusion: Anatomic regions of the glenoid labrum differ in extracellular matrix composition, vascularity, and cell composition. The superior glenoid labrum is deficient in vascularity and progenitor cells, which may explain the high failure rates for repairs in this location. Clinical Relevance: Improved understanding of the composition of distinct glenoid labral positions may help to improve therapeutic strategies for labral pathology.

Abstract 364: Vascular Endothelial Growth Receptor 2 (Flk1) Donor Exosomes Drive Angiogenesis

Objectives: Peripheral arterial disease is a significant cause of morbidity and mortality. The goal of this study was to develop a novel treatment to promote angiogenesis. Platelets have been previously implicated in angiogenic events; however, platelet activation rather than presence of growth factors has been associated with new blood vessel formation. This study evaluated exosomes purified from activated platelets to determine their potential in promoting angiogenesis both in vitro and in vivo. Methods &amp; Results: Under Current Good Manufacturing Practices, conditioned medium processed from apheresis platelet cultures yielded a purified exosome product (PEP) enriched for CD63, CD9 and Alix. In vitro, PEP drove HUVEC proliferation, migration, and vascular tube formation. Proteomic evaluation showed Flk1 (VEGFR2) was one of the key angiogenic proteins in PEP. HUVECs treated with Flk1 antibody neutralized PEP (anti-Flk1-PEP) showed reduced vascular tube formation compared to PEP. To evaluate in vivo angiogenesis, a rodent model of hindlimb ischemia was treated with sustained release PEP embedded in fibrin glue (PEP/TISSEEL) with blood flow evaluated by SPY angiography at 28 days post-treatment. Animals treated with PEP/TISSEEL showed improved blood flow with significant difference (p&lt;0.001) as compared to sham, TISSEEL, and anti-Flk1-PEP/TISSEEL. Muscle tissue stained with α smooth muscle actin showed a significant increase (p&lt;0.001) in vascular area in PEP/TISSEEL treated animals compared to TISSEEL alone. To further evaluate clinical translatability, a rabbit ear ischemia wound model was then treated with PEP/TISSEEL. Rabbit ears showed accelerated wound healing and restored blood flow on SPY angiography. Histologic evaluation of vascular area showed a significant increase in vascular area (p&lt;0.05) compared to sham and TISSEEL alone animals. Conclusions: Exosome driven donation of Flk1 here promoted angiogenic events and was successfully translated into healing outcomes in the setting of two animal models of peripheral ischemia. This novel insight into biotherapy achievable pro-angiogenesis establishes a clinical-grade off-the-shelf exosome technology for the treatment of peripheral vascular disease.

Human ex vivo model of iris angiogenesis

PurposeTo assess iris angiogenesis using human explants and determine the role of hypoxia during iris neovascularization.MethodsHuman irises were isolated post‐mortem from human research consented donnors. Explants were seeded onto extracellular matrix (ECM), and kept at normal oxygen concentrations (normoxia, 20% O2) or exposed to hypoxia (1% O2) in standard tissue culture conditions for 48 h. Iris angiogenesis was analyzed by quantifying the neovascular area. Furthermore, iris neovascular structures were characterized by immunofluorescence.ResultsHuman iris ex vivo neoangiogenesis could be observed within 48 h after the explants were seeded onto ECM, and was defined as vascular sprouts and/or tufts. Vascular sprouts were more common in normoxia, while vascular tufts were mostly formed in hypoxia. A significant increase in vascular area was observed in irises exposed to hypoxia when compared to normoxia. Positive immunoreactivity to specific endothelial cell markers confirmed human iris neoangiogenesis ex vivo.ConclusionsOur data shows the first human ex vivo neoangiogenesis assay, using iris. The method here presented could have potential use in testing pro‐ and anti‐angiogenic drugs in an human ocular context.

Apoptose e maturação placentária bovina: um estudo imunohistoquímico e morfométrico

Resumo: A liberação da placenta após o parto envolve a perda da adesão materno-fetal e ocorre somente após a maturação completa do placentoma, que está relacionada com a diminuição da celularidade dos tecidos fetal e materno. A apoptose é requerida tanto para a maturação quanto para a liberação normal da placenta após o parto. O objetivo do presente estudo foi avaliar a ocorrência de apoptose em amostras de placenta de vacas em diferentes fases de gestação. Amostras de placentomas de 15 vacas saudáveis com 4 (n=5), 6 (n=5) e 9 (n=5) meses de gestação foram colhidas e processadas rotineiramente para a histologia, imunoistoquímica e histoquímica. As lâminas obtidas foram coradas em HE, Picrosirius Red e submetidas à análise imunoistoquímica das proteínas Caspase 3, Caspase 8, Bax e Bid. O aumento no número de vasos não necessariamente se associou ao aumento do calibre destes durante a evolução da gestação. Os resultados de histomorfometria revelaram aumento da marcação para Bax e Caspases 3 e 8 em células trofoblásticas binucleadas no final da gestação, enquanto o Bid se manteve sem alteração significativa. A histomorfometria das células trofoblásticas mononucleadas revelou expressão alta para Bax no início de gestação, com diminuição aos 6 meses de gestação e aumento das imunomarcações para Caspases 3 e 8, e Bid com o avanço gestacional. Os colágenos tipo I e III não aumentaram do terço médio ao final da gestação, o que é importante para a diminuição da adesão materno-fetal. Esses resultados confirmam que as Caspases 3 e 8, e o Bax estão envolvidos nos mecanismos de ativação da apoptose pela via intrínseca mitocondrial e/ou extrínseca ao longo da gestação em células trofoblásticas binucleadas, e que nas células trofoblásticas mononucleadas o Bax deixa de ser importante, enquanto o Bid e as Caspases 3 e 8 se tornam os mais significativos.

Increased vascular permeability in the circumventricular organs of adult rat brain due to stimulation by extremely low frequency magnetic fields

It has been demonstrated that the exposure of biological systems to magnetic fields (MFs) can produce several beneficial effects: tissue recovery in chronic wounds, re-establishment of blood circulation after tissue ischemia or in necrotic tissues, improvement after epileptic episodes, angiogenesis, etc. In the current study, the effects of extremely low frequency (ELF) MF on the capillaries of some circumventricular organs (CVOs) are demonstrated; a vasodilator effect is reported as well as an increase in their permeability to non-liposoluble substances. For this study, 96 Wistar male rats (250 g body mass) were used and divided into three groups of 32 rats each: a control group (no treatment); a sham ELF-MF group; and an experimental group subjected to ELF-MF (120 Hz harmonic waves and 0.66 mT, root mean square) by the use of Helmholtz coils. All animals were administered colloidal carbon (CC) intravenously to study, through optical and transmission electron microscopy, the capillary permeability in CVOs and the blood-brain barrier (BBB) in brain areas. An increase in capillary permeability to CC was detected in the ELF-MF-exposed group as well as a significant increase in vascular area (capillary vasodilation); none of these effects were observed in individuals of the control and sham ELF-MF groups. It is important to investigate the mechanisms involved in the phenomena reported here in order to explain the effects of ELF-MF on brain vasculature.

Spontaneously hypertensive rat mesenteric microvsascular networks undergo angiogenesis in response to a wound healing stimulus

Hypertension is associated with increased microvascular resistance due in part to a loss of microvessels, termed microvascular rarefaction. Understanding whether this loss of microvessels is a result of vessel regression or impaired growth requires further investigation of the ability of hypertensive networks to undergo angiogenesis. The objective of this study was to determine the angiogenic response of mesenteric microvascular networks in adult spontaneously hypertensive versus normotensive rats. Mesenteric tissues were harvested from 15‐16 week old spontaneously hypertensive and Wistar rats according to 3 experimental groups: unstimulated, 3 days post and 5 days post wound healing stimulus induced by 20 min exteriorization of the mesentery. Microvascular networks were immunohistochemically identified by PECAM labeling. Consistent with the phenomenon of rarefaction, SHR unstimulated microvascular networks were smaller than normotensive controls. By 3 days post stimulation both SHR and Wistar microvascular networks displayed increased capillary sprouting. By 5 days both SHR and Wistar microvascular networks displayed an increase in vascular area. These observations suggest that hypertensive microvascular networks are able to undergo angiogenesis similar to normotensive networks.

Subepithelial vascular patterns in bronchial dysplasias using a high magnification bronchovideoscope

Background: We have developed a method of high magnification bronchovideoscopy that enables improved observation of subepithelial vascular patterns of the bronchial mucosa. A study was undertaken to investigate the value...

Pathogenesis of Classical Swine Fever: Renal Haemorrhages and Erythrodiapedesis

Thirty pigs were inoculated with a virulent isolate (Quillota strain) of classical swine fever (hog cholera) virus to establish the chronological occurrence of lesions in the kidney and to determine the mechanism responsible for renal haemorrhages. The study included the use of histopathological, ultrastructural, immunohistochemical (detection of viral antigen gp55, MAC387, λ chains, CD3 and C1q) and morphometrical techniques (vascular area). Renal interstitial oedema and haemorrhages were detected from 7 days post-inoculation (dpi), associated with a slight interstitial mononuclear infiltrate and evidence of viral infection in macrophages and fibroblasts, and in a small proportion of lymphocytes. Viral infection was not detected in capillary endothelial cells. An intense mononuclear infiltrate, with B cells, T cells and small numbers of macrophages, was detected from 10 dpi. In the final phase of the experiment (14 dpi), slight proliferation and degranulation of mast cells were observed. Increased expression of the C1q component of complement was also detected. A significant increase in vascular area was observed from 7 dpi. These results suggest that haemorrhages observed in the kidneys of pigs inoculated with the Quillota strain resulted from erythrodiapedesis and increased vascular permeability, probably aggravated by mast cell degranulation in the final stage of the experiment. The results suggested that mast cell degranulation was linked to activation of the complement system.

Effects of bronchial vascular engorgement on airway dimensions.

Airway vascular engorgement has been suggested to cause luminal narrowing and airflow obstruction. To determine the extent to which changes in bronchial vascular volume could influence airway dimensions, we studied the effects of left atrial pressure elevation on airway morphometry in sheep (n = 17). The bronchial branch of the bronchoesophageal artery was cannulated and perfused with autologous blood (0.6 ml.min-1.kg-1). A balloon-tipped catheter was inserted into the left atrial appendage to elevate left atrial pressure by 10 mmHg, and papaverine was infused into the bronchial artery to eliminate airway smooth muscle tone. Morphological measurements were made from rapidly frozen lungs excised in vivo. Left atrial pressure elevation caused a 79% increase in total vascular area (P = 0.0002). Average airway luminal area was significantly decreased from 86 to 71% of the airway maximal area (P < 0.0001). Noteworthy were the prominent bronchial vessels located within mucosal folds. However, when papaverine was infused during left atrial pressure elevation, despite a comparable total vascular area, luminal narrowing did not occur and remained at 87% of the maximal area (P = 0.6267). In conclusion, we found that engorgement of the bronchial vasculature leads to an increase in the vascular area in regions inside and outside the smooth muscle layer. The associated decrease in luminal area only occurs in the presence of airway smooth muscle tone. This suggests a reflex effect on the airway caused by the vascular engorgement. We conclude that vascular engorgement of the airway wall per se has a negligible effect on airway obstruction.