Randomized Study Comparing the Effect of Carbon Dioxide Insufflation on Veins Using 2 Types of Endoscopic and Open Vein Harvesting.

OBJECTIVE: The aim of the study was to assess whether the use of carbon dioxide insufflation has any impact on integrity of long saphenous vein comparing 2 types of endoscopic vein harvesting and traditional open vein harvesting. METHODS: A total of 301 patients were prospectively randomized into 3 groups. Group 1 control arm of open vein harvesting (n = 101), group 2 closed tunnel (carbon dioxide) endoscopic vein harvesting (n = 100) and Group 3 open tunnel (carbon dioxide) endoscopic vein harvesting (open tunnel endoscopic vein harvesting) (n = 100). Each group was assessed to determine the systemic level of partial arterial carbon dioxide, end-tidal carbon dioxide, and pH. Three blood samples were obtained at baseline, 10 minutes after start of endoscopic vein harvesting, and 10 minutes after the vein was retrieved. Vein samples were taken immediately after vein harvesting without further surgical handling to measure the histological level of endothelial damage. A modified validated endothelial scoring system was used to compare the extent of endothelial stretching and detachment. RESULTS: The level of end-tidal carbon dioxide was maintained in the open tunnel endoscopic vein harvesting and open vein harvesting groups but increased significantly in the closed tunnel endoscopic vein harvesting group (P = 0.451, P = 0.385, and P < 0.001). Interestingly, partial arterial carbon dioxide also did not differ over time in the open tunnel endoscopic vein harvesting group (P = 0.241), whereas partial arterial carbon dioxide reduced significantly over time in the open vein harvesting group (P = 0.001). A profound increase in partial arterial carbon dioxide was observed in the closed tunnel endoscopic vein harvesting group (P < 0.001). Consistent with these patterns, only the closed tunnel endoscopic vein harvesting group demonstrated a sudden drop in pH over time (P < 0.001), whereas pH remained stable for both open tunnel endoscopic vein harvesting and open vein harvesting groups (P = 0.105 and P = 0.869, respectively). Endothelial integrity was better preserved in the open vein harvesting group compared with open tunnel endoscopic vein harvesting or closed tunnel endoscopic vein harvesting groups (P = 0.012) and was not affected by changes in carbon dioxide or low pH. Significantly greater stretching of the endothelium was observed in the open tunnel endoscopic open tunnel endoscopic vein harvesting group compared with the other groups (P = 0.003). CONCLUSIONS: This study demonstrated that the different vein harvesting techniques impact on endothelial integrity; however, this does not seem to be related to the increase in systemic absorption of carbon dioxide or to the pressurized endoscopic tunnel. The open tunnel endoscopic harvesting technique vein had more endothelial stretching compared with the closed tunnel endoscopic technique; this may be due to manual dissection of the vein. Further research is required to evaluate the long-term clinical outcome of these vein grafts.

Evaluation of the effects of intra-arterial sugammadex and dexmedetomidine: an experimental study / Avaliação dos efeitos de sugamadex e dexmedetomidina intra-arterial: estudo experimental

Abstract Background: Intra-arterial injection of medications may cause acute and severe ischemia and result in morbidity and mortality. There is no information in the literature evaluating the arterial endothelial effects of sugammadex and dexmedetomidine. The hypothesis of our study is that sugammadex and dexmedetomidine will cause histological changes in arterial endothelial structure when administered intra-arterially. Methods: Rabbits were randomly divided into 4 groups. Group Control (n = 7); no intervention performed. Group Catheter (n = 7); a cannula inserted in the central artery of the ear, no medication was administered. Group Sugammadex (n = 7); rabbits were given 4 mg/kg sugammadex into the central artery of the ear, and Group Dexmedetomidine (n = 7); rabbits were given 1 µg/kg dexmedetomidine into the central artery of the ear. After 72 h, the ears were amputated and histologically investigated. Results: There was no significant difference found between the control and catheter groups in histological scores. The endothelial damage, elastic membrane and elastic fiber damage, smooth muscle hypertrophy and connective tissue increase scores in the dexmedetomidine and sugammadex groups were significantly higher than both the control and the catheter groups (p < 0.05). There was no significant difference found between the dexmedetomidine and sugammadex groups in histological scores. Conclusion: Administration of sugammadex and dexmedetomidine to rabbits by intra-arterial routes caused histological arterial damage. To understand the histological changes caused by sugammadex and dexmedetomidine more clearly, more experimental research is needed.

Resumo Justificativa: A injeção intra-arterial de medicamentos pode causar isquemia aguda e grave e resultar em morbidade e mortalidade. Não há informações na literatura que avaliem os efeitos endoteliais arteriais de sugamadex e dexmedetomidina. A hipótese de nosso estudo foi que dexmedetomidina e sugamadex causariam alterações histológicas na estrutura endotelial arterial quando administrados por via intra-arterial. Método: Os coelhos foram randomicamente divididos em quatro grupos: grupo controle (n = 7), sem intervenção; grupo cateter (n = 7), uma cânula foi inserida na artéria central da orelha e medicamentos não foram administrados; grupo sugamadex (n = 7), receberam 4 mg/kg de sugamadex na artéria central da orelha; grupo dexmedetomidina (n = 7), receberam 1 µg/kg de dexmedetomidina na artéria central da orelha. Após 72 horas, as orelhas foram amputadas e histologicamente examinadas. Resultados: Não houve diferença significativa entre os grupos controle e cateter referente aos escores histológicos. Os escores do dano causado ao endotélio e à membrana e fibra elásticas, da hipertrofia do músculo liso e do aumento do tecido conjuntivo foram significativamente maiores nos grupos dexmedetomidina e sugamadex do que nos grupos controle e cateter (p < 0,05). Não houve diferença significativa entre os grupos dexmedetomidina e sugamadex nos escores histológicos. Conclusão: A administração de sugamadex e dexmedetomidina a coelhos por via intra-arterial causou danos arteriais histológicos. Para entender as alterações histológicas causadas por sugamadex e dexmedetomidina com mais clareza, estudos experimentais adicionais são necessários.

Intravital imaging of embryonic and tumor neovasculature using viral nanoparticles.

Viral nanoparticles are a novel class of biomolecular agents that take advantage of the natural circulatory and targeting properties of viruses to allow the development of therapeutics, vaccines and imaging tools. We have developed a multivalent nanoparticle platform based on the cowpea mosaic virus (CPMV) that facilitates particle labeling at high density with fluorescent dyes and other functional groups. Compared with other technologies, CPMV-based viral nanoparticles are particularly suited for long-term intravital vascular imaging because of their biocompatibility and retention in the endothelium with minimal side effects. The stable, long-term labeling of the endothelium allows the identification of vasculature undergoing active remodeling in real time. In this study, we describe the synthesis, purification and fluorescent labeling of CPMV nanoparticles, along with their use for imaging of vascular structure and for intravital vascular mapping in developmental and tumor angiogenesis models. Dye-labeled viral nanoparticles can be synthesized and purified in a single day, and imaging studies can be conducted over hours, days or weeks, depending on the application.

Regulation of vascular tone and remodeling of the ductus arteriosus.

The ductus arteriosus (DA), a fetal arterial connection between the main pulmonary artery and the descending aorta, normally closes immediately after birth. The DA is a normal and essential fetal structure. However, it becomes abnormal if it remains patent after birth. Closure of the DA occurs in two phases: functional closure of the lumen within the first hours after birth by smooth muscle constriction, and anatomic occlusion of the lumen over the next several days due to extensive neointimal thickening in human DA. There are several events that promote the DA constriction immediately after birth: (a) an increase in arterial oxygen tension, (b) a dramatic decline in circulating prostaglandinE(2) (PGE(2)), (c) a decrease in blood pressure within the DA lumen, and (d) a decrease in the number of PGE(2) receptors in the DA wall. Anatomical closure of the DA is associated with the formation of intimal thickening, which are characterized by (a) an area of subendothelial deposition of extracellular matrix, (b) the disassembly of the internal elastic lamina and loss of elastic fiber in the medial layer, and (c) migration into the subendothelial space of undifferentiated medial smooth muscle cells. In addition to the well-known vasodilatory role of PGE(2), our findings uncovered the role of PGE(2) in anatomical closure of the DA. Chronic PGE(2)-EP4-cyclic AMP (cAMP)-protein kinase A (PKA) signaling during gestation induces vascular remodeling of the DA to promote hyaluronan-mediated intimal thickening and structural closure of the vascular lumen. A novel target of cAMP, Epac, has an acute promoting effect on smooth muscle cell migration without hyaluronan production and thus intimal thickening in the DA. Both EP4-cAMP downstream targets, Epac and PKA, regulate vascular remodeling in the DA.

Postnatal maturation of tendon, cruciate ligament, meniscus and articular cartilage: a histological study in sheep.

Orthopaedic basic science data on immature skeletons are rare in the literature. Since the number of knee injuries in young humans is steadily increasing, studies on immature animals such as sheep, which can be used as model systems are becoming more and more important. However, no baseline data are available on physiologic and morphologic changes during growth in the relevant tissues. In the present study, histomorphometric changes in the tendon of the musculus flexor digitalis superficialis, the cranial cruciate ligament, the medial meniscus and the articular cartilage of the medial femoral condyle were identified in sheep between the ages of 1 and 40 weeks postnatally. Profound changes in tissue composition during growth could be observed. A high cellularity in the early postnatal period decreases to a constant lower level after 18 weeks. Similar changes during postnatal growth could be observed for blood vessel density. Also, staining of alpha-smooth muscle actin (SMA) and vascular endothelial growth factor (VEGF) steadily decreased. In contrast, the number of components of extracellular matrix steadily increased in all tissues. The age of 18 weeks seems to be a threshold after which the tissue composition of the observed structures remains constant in this species.

Spatial and temporal role of the apelin/APJ system in the caliber size regulation of blood vessels during angiogenesis.

Blood vessels change their caliber to adapt to the demands of tissues or organs for oxygen and nutrients. This event is mainly organized at the capillary level and requires a size-sensing mechanism. However, the molecular regulatory mechanism involved in caliber size modification in blood vessels is not clear. Here we show that apelin, a protein secreted from endothelial cells under the activation of Tie2 receptor tyrosine kinase on endothelial cells, plays a role in the regulation of caliber size of blood vessel through its cognate receptor APJ, which is expressed on endothelial cells. During early embryogenesis, APJ is expressed on endothelial cells of the new blood vessels sprouted from the dorsal aorta, but not on pre-existing endothelial cells of the dorsal aorta. Apelin-deficient mice showed narrow blood vessels in intersomitic vessels during embryogenesis. Apelin enhanced endothelial cell proliferation in the presence of vascular endothelial growth factor and promoted cell-to-cell aggregation. These results indicated that the apelin/APJ system is involved in the regulation of blood vessel diameter during angiogenesis.

Experiencia clínica del interferón alfa 2B en hemangioma periorbitario: a propósito de un caso / Clinical experience of the interferon alpha 2B in hemangioma periorbitario: a purpose of a case

Los hemangiomas son tumores de partes blandas, del endotelio vascular y benigno más frecuente en la infancia, con una incidencia entre 5 por ciento y 15 por ciento. Se consideran también los tumores primarios más frecuentes de la órbita. En este estudio, se recoge nuestra experiencia con interferón alfa en el tratamiento de hemangiomas orbitarios infantiles. Se estudia la eficacia del mismo y la aparición de efectos secundarios atribuibles al tratamiento. Preescolar masculino de 28 meses de edad, quien desde los 12 meses presentaba discreto aumento de volumen en parpado superior derecho y de dos meses y medio de evolución aumento progresivo, cambios de coloración y desplazamiento del globo ocular hacia fuera de la línea media. Valorado por los Servicios de Oftalmología y Oncología Pediátrica deciden iniciar protocolo de tratamiento con interferón alfa 2B. El paciente recibió el fármaco durante 2 meses y medio consecutivos tres veces por semana, vía subcutánea, a 3.000.000 Uds/m2SC/dosis; encontrándose efectos adversos, durante los primeros días de la administración; los últimos 15 días el tratamiento se cumplió diariamente sin efectos adversos. Al cabo de los tres meses se observo reorganización del hemangioma con aplanamiento de la coloración violácea y disminución de 1cm de diámetro. La incidencia de complicaciones de los hemangiomas periorbitarios es muy alta. Según diferentes autores, el interferón alfa, consigue una reducción de más del 50 por ciento del tamaño del hemangioma en 58 por ciento de los casos, y en 26 por ciento una reducción entre el 20 y 40 por ciento. En aproximadamente un 15 por ciento de los casos, no existe respuesta al tratamiento.

Use of immunohistochemical stains to assess the variations in color of fat of the upper eyelid.

Although there are many variations on the technique, most practitioners agree that removal of periorbital eyelid fat is an integral part of the blepharoplasty operation. The retrieval and excision of the medial fat of the upper eyelid is a step that, when overlooked, leaves the patient with an unsatisfactory appearance of puffiness in this area. The medial fat is characteristically white; the other fat compartments are yellow. The authors attempt to understand why these two juxtaposed fat pockets are of distinct different colors. Four consecutive patients underwent traditional upper blepharoplasty surgery. From each patient two separate fat specimens were identified as upper lateral and upper medial. These fat specimens were processed with hematoxylin and eosin and various immunohistochemical stains. A board-certified pathologist, who was blinded as to the anatomic site of the fat, evaluated them. The medial fat showed much larger fat lobules than the upper lateral fat. Contrasting this, the lateral fat had many more endothelial-lined blood vessels and much thicker fibrous septa than its medial neighbor. These findings were supported immunohistochemically by using CD34 (endothelial antibody) and factor XIIIa (dermal dendritic cell/ normal fibroblast antibody). The use of other immunostains using various antibodies did not distinguish any distinct histomorphology among the specimens. Although for the operating surgeon it is important to recognize the white color of the medial fat to aid in identification during surgery, one can only speculate as to what contributes to this difference in blood vessels and supporting fibrous septa. Most likely this is the result of anatomic, embryologic, and vascular bed differences.

Early adaptive responses of the vascular wall during venous arterialization in mice.

Venous arterialization occurs when a vein segment is transposed as a bypass graft into the arterial circulation, resulting in a structural and functional reorganization of the vascular wall in response to the new local biomechanical environment. Although the anatomical changes of venous arterialization have been well characterized, the molecular mechanisms of vascular remodeling remain incompletely understood. Here, we present a novel model of venous arterialization in mice wherein the external jugular vein is connected to the common carotid artery. The hemodynamic characteristics of the arterialized vein, as assessed by ultrasound and magnetic resonance imaging, resemble features of the arterial circulation. Temporal analyses of the morphological changes in the venous segment at 1, 3, and 7 days after surgery demonstrate preservation of the endothelium at all time points and formation of multiple smooth muscle layers by day 7. Expression of endothelial E-selectin and VCAM-1 was documented at early time points, concomitant with the presence of neutrophils and monocytes/macrophages in the vascular wall. In addition, endothelium-dependent permeability was decreased in the arterialized vein when compared to the contralateral control vein. Thus, this novel mouse model of venous arterialization displays anatomical and cellular features present in other species, and should help to characterize the molecular mechanisms of this adaptive response of the vascular wall to changes in its biomechanical environment.

Initial angiogenic response in reduced renal mass after transplantation.

INTRODUCTION: Shortage of organs is a major problem in kidney transplantation and requires novel strategies to increase the number of kidney transplants. To reduce the shortage of kidneys, we have proposed transplantation of two halves of one kidney into two recipients (hemirenal transplantation, HRT) and have shown its feasibility in pig and human kidneys. However, reduced renal mass can lead to progressive renal failure in rodents and can reduce the longevity of kidney transplants in humans. Recent studies suggest that derangement of angiogenesis plays a role in the progressive renal failure after reduction in renal mass in rodents. However, since the renal physiology of rats is different from that of large animals, we studied angiogenesis in reduced renal mass transplants in pigs and determined if the reduction in renal mass has the same effect in large animals as that in rodents. MATERIALS AND METHODS: Kidney autotransplantation was performed in domestic outbred swine. Heminephrectomy of the autotransplanted kidney and nephrectomy of the contralateral kidney were performed 1 week after transplantation to reduce the renal mass. Four weeks after transplantation, the pigs were sacrificed and the hemirenal and control nephrectomy specimens were processed for morphometric analysis of glomerular capillary density and immunohistochemical analysis of VEGF expression. Soluble extracts from the kidneys were tested in an in vitro angiogenesis assay to determine their activity to influence angiogenesis. Statistical analysis with ANOVA was performed on the glomerular capillary density in kidney specimens. RESULTS: All these parameters of angiogenesis were increased in the reduced renal mass autotransplants as compared to normal kidneys or whole kidney autotransplants. Glomerular capillary density was increased significantly after reduction in renal mass. VEGF expression also was increased progressively by the third week after reduction in renal mass. Soluble extract from the reduced renal mass transplants significantly increased the in vitro angiogenesis. CONCLUSION: This is the first study to demonstrate that angiogenesis is increased in the initial stages of reduction in renal mass after transplantation in a large animal model. Increased angiogenesis was found in this model earlier than reported in small animal models (2 weeks in pigs versus 6 weeks in rats). Taken together with other studies, our data suggest that derangement in angiogenesis could play an important role in long-term graft function after hemirenal transplantation.

Coordinated functions of Akt/PKB and ETS1 in tubule formation.

We investigated the inter-relationship between two downstream effectors of vascular endothelial growth factor (VEGF), the serine threonine kinase Akt (also known as protein kinase B) and the transcription factor ETS1, during tubulogenesis. Human endothelial cell culture and the in vivo Drosophila tracheal systems are employed in comparative analysis. We show that VEGF stimulates the expression of ETS1 through a phosphatidylinositol-3-kinase (PI3K)/Akt-dependent pathway in primary endothelial cells. Activation of Akt results in vessel formation in vitro, a process that is blocked by expression of antisense ETS1. The functional relationship between ETS and Akt was then tested in the homologous tubular system in Drosophila. Contrary to expectation, ETS1 and Akt did not form a linear positive regulatory pathway in vivo. Instead, genetic analyses suggest that the Drosophila ETS1 homologue Pointed is required for cell motility per se while Drosophila Akt (Dakt1) is responsible for organized and restricted cell movement that is essential for tubule formation. Taken together, our results show that ETS1 and Akt control different aspects of cell motility that are integrated in the precise regulation of vascular tubule formation.

First evidence that bone marrow cells contribute to the construction of tissue-engineered vascular autografts in vivo.

BACKGROUND: Materials commonly used to repair complex cardiac defects lack growth potential and have other unwanted side effects. We designed and tested a bone marrow cell (BMC)-seeded biodegradable scaffold that avoids these problems. METHODS AND RESULTS: To demonstrate the contribution of the BMCs to histogenesis, we labeled them with green fluorescence, seeded them onto scaffolds, and implanted them in the inferior vena cava of dogs. The implanted grafts were analyzed immunohistochemically at 3 hours and subsequently at 2, 4, and 8 weeks after implantation using antibodies against endothelial cell lineage markers, endothelium, and smooth muscle cells. There was no stenosis or obstruction caused by the tissue-engineered vascular autografts (TEVAs) implanted into the dogs. Immunohistochemically, the seeded BMCs expressing endothelial cell lineage markers, such as CD34, CD31, Flk-1, and Tie-2, adhered to the scaffold. This was followed by proliferation and differentiation, resulting in expression of endothelial cells markers, such as CD146, factor VIII, and CD31, and smooth muscle cell markers, such as alpha-smooth muscle cell actin, SMemb, SM1, and SM2. Vascular endothelial growth factor and angiopoietin-1 were also produced by cells in TEVAs. CONCLUSIONS: These results provide direct evidence that the use of BMCs enables the establishment of TEVAs. These TEVAs are useful for cardiovascular surgery in humans and especially in children, who require biocompatible materials with growth potential, which might reduce the instance of complications caused by incompatible materials and lead to a reduced likelihood of further surgery.

Mapping of vascular endothelium in the human flexor digitorum profundus tendon.

PURPOSE: Previous techniques to delineate the human flexor digitorum profundus (FDP) vasculature have been innovative but potentially imprecise, resulting in uncertainty as to the existence of avascular zones in the flexor tendon. We aimed to use a novel immunohistochemical technique to determine more accurately the vasculature of the human flexor tendon. METHODS: Thirty fresh cadaveric human FDP tendons were harvested, fixed, wax embedded, sectioned, and stained using the anti-CD31 monoclonal antibody to allow vessel visualization. Vessel numbers and vascularity density ratios were determined by computed image analysis. RESULTS: Vessel density ratios varied with anatomic location, with a decrease between the A2 and A4 pulleys. There also was variation in vascularity ratios in the anteroposterior plane, with the palmar surface of the tendons having a lower vessel density. CONCLUSIONS: We have shown that although areas of low vascularity exist on the palmar aspect of the tendon, there are no truly avascular zones.

Angiogenic role of adrenomedullin through activation of Akt, mitogen-activated protein kinase, and focal adhesion kinase in endothelial cells.

Adrenomedullin (AM) is a multifunctional peptide in human pheochromocytoma. To evaluate whether AM could be an angiogenic factor, we examined its effect on kinases and angiogenic processes. AM induced tyrosine phosphorylation of Akt and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase1/2 (ERK1/2) by using distinct signaling pathways in human umbilical vein endothelial cells (HUVECs). AM also phosphorylated focal adhesion kinase, and phosphatidylinositol 3'-kinase inhibitor inhibited AM-induced focal adhesion kinase phosphorylation. Pretreatment with high concentrations of AM22-52, a putative AM receptor antagonist, partially suppressed AM-induced phosphorylation of Akt, ERK1/2, and focal adhesion kinase. AM and vascular endothelial growth factor produced increases in DNA synthesis and migration in HUVECs. AM induced tube formation in HUVECs, and its effect was inhibited by pretreatment with phosphatidylinositol 3'-kinase inhibitor or ERK1/2 inhibitor. AM induced sprouting in porcine pulmonary arterial endothelial cells and promoted neovessel formation in a mouse Matrigel plug assay. Inhibitors of phosphatidylinositol 3'-kinase and ERK1/2 inhibited AM-induced endothelial sprouting in vitro and angiogenesis in vivo. AM exerts angiogenic activity through activation of Akt, MAPK, and focal adhesion kinase in endothelial cells.

Molecular regulation of vessel maturation.

The maturation of nascent vasculature, formed by vasculogenesis or angiogenesis, requires recruitment of mural cells, generation of an extracellular matrix and specialization of the vessel wall for structural support and regulation of vessel function. In addition, the vascular network must be organized so that all the parenchymal cells receive adequate nutrients. All of these processes are orchestrated by physical forces as well as by a constellation of ligands and receptors whose spatio-temporal patterns of expression and concentration are tightly regulated. Inappropriate levels of these physical forces or molecules produce an abnormal vasculature--a hallmark of various pathologies. Normalization of the abnormal vasculature can facilitate drug delivery to tumors and formation of a mature vasculature can help realize the promise of therapeutic angiogenesis and tissue engineering.

Angiostatin and endostatin inhibit endothelial cell migration in response to FGF and VEGF without interfering with specific intracellular signal transduction pathways.

The anti-angiogenic agents angiostatin and endostatin have been shown to affect endothelial cell migration in a number of studies. We have examined the effect of these agents on intracellular signalling pathways known to regulate endothelial cell migration and proliferation/survival. Both agents inhibited fibroblast growth factor (FGF)-, and vascular endothelial growth factor (VEGF)-mediated migration of primary human microvascular endothelial cells and affected vascular formation in the embryoid body model. However, using phosphospecific antibodies we could not detect any effect of angiostatin or endostatin on phospholipase C-gamma (PLC-gamma), Akt/PKB, p44/42 mitogen-activated protein kinase (MAPK), p38 MAPK and p21-activated kinase (PAK) activity. Furthermore, using a glutathione S-transferase (GST)-PAK pull-down assay, we could not detect any effect on Rac activity. We conclude that angiostatin and endostatin inhibit chemotaxis, without affecting intracellular signalling pathways known to regulate endothelial migration and proliferation/survival.

Angiogenesis of the blood-brain barrier in vitro and the function of cerebral pericytes.

Cerebral pericytes constitute an essential component of the blood-brain barrier (BBB) and are involved in blood vessel assembly. Recently, we reported on the induction of a BBB-specific enzyme expressed by cerebral pericytes (pericytic aminopeptidase N/pAPN) in coculture with cerebral endothelial cells. We completed this in vitro BBB system by adding astrocytes to these mixed cultures of endothelial cells and pericytes. Under these triculture conditions, endothelial cells and pericytes reorganize into capillary-like structures (CLSs). Capillary formation can also be achieved by the application of transforming growth factor beta 1 (TGF-b1) in the culture medium of endothelial-pericyte cultures lacking astrocytes. In contrast to the effect achieved by astrocytes, pericytes did not assemble with endothelial cells. In both cases (application of astrocytes or TGF-b1), endothelial cells underwent apoptosis. However, endothelial cells that form CLSs in the presence of pericytes appeared to be resistant to induction of apoptosis. On the basis of these observations, we concluded that astrocytes have a profound influence on the morphogenetic events underlying the organization of the vessel wall; that the effect of TGF-b1 is different from the astrocytic effect because it lacks induction of endothelial-pericyte association; and that pericytes stabilize CLSs formed by endothelial cells in coculture with astrocytes.

The Cdc42 and Rac1 GTPases are required for capillary lumen formation in three-dimensional extracellular matrices.

Here we show a requirement for the Cdc42 and Rac1 GTPases in endothelial cell (EC) morphogenesis in three-dimensional extracellular matrices. Cdc42 and Rac1 specifically regulate EC intracellular vacuole and lumen formation in both collagen and fibrin matrices. Clostridium difficile toxin B (which blocks all three Rho GTPases) completely inhibited the ability of ECs to form both vacuoles and lumens, whereas C3 transferase, a selective inhibitor of Rho, did not. Expression of either dominant-negative (N17) or constitutively active (V12) Cdc42 using recombinant adenoviruses dramatically inhibited EC vacuole and lumen formation in both collagen and fibrin matrices. Both vacuole and lumen formation initiated in ECs expressing dominant-negative (N17) Rac1 but later collapsed, indicating a role for Rac1 during later stages of vessel development. Analysis of cultures using confocal microscopy revealed green fluorescent protein-V12Rac1, -Rac1 wild-type and -Cdc42 wild-type chimeric proteins targeted to intracellular vacuole membranes during the lumen formation process. Also, expression of the verprolin-cofilin-acidic domain of N-WASP, a downstream Cdc42 effector, in ECs completely interfered with vacuole and lumen formation. These results collectively reveal a novel role for Cdc42 and Rac1 in the process of EC vacuole and lumen formation in three-dimensional extracellular matrices.

Fibrocytes induce an angiogenic phenotype in cultured endothelial cells and promote angiogenesis in vivo.

Angiogenesis is an ordered process requiring the inter-play of numerous cellular and humoral factors. Studies over the past 20 years have identified several growth factors, cytokines, and enzymes that promote blood vessel formation. Most have revealed how individual factors promote an angiogenic phenotype in endothelial cells in vitro or contribute to blood vessel formation in vivo. However, the fundamental question that remains unanswered is how the cellular microenvironment contributes to angiogenesis. Fibrocytes are a recently characterized mesenchymal cell type isolated from peripheral blood that rapidly enter subcutaneously implanted wound chambers and sites of tissue injury. Here we describe the induction of an angiogenic phenotype in microvascular endothelial cells in vitro and promotion of angiogenesis in vivo by cultured fibrocytes. Fibrocytes constitutively secrete extracellular matrix-degrading enzymes, primarily matrix metalloproteinase 9, which promotes endothelial cell invasion. In addition, fibrocytes secrete several proangiogenic factors including VEGF, bFGF, IL-8, PDGF, and hematopoietic growth factors that promote endothelial cell migration, proliferation, and/or tube formation. By contrast, they do not produce representative antiangiogenic factors. Finally, both autologous fibrocytes and fibrocyte-conditioned media were found to induce blood vessel formation in vivo using the Matrigel angiogenesis model.