Michael Bernas

Defective remodeling and maturation of the lymphatic vasculature in Angiopoietin-2 deficient mice

Molecular mechanisms regulating the remodeling of the lymphatic vasculature from an immature plexus of vessels to a hierarchal network of initial and collecting lymphatics are not well understood. One gene thought to be important for this process is Angiopoietin-2 (Ang-2). Ang2(-/-) mice have previously been reported to exhibit an abnormal lymphatic phenotype but the precise nature of the lymphatic defects and the underlying mechanisms have yet to be defined. Here we demonstrate by whole-mount immunofluorescence staining of ear skin and mesentery that lymphatic vessels in Ang2(-/-) mice fail to mature and do not exhibit a collecting vessel phenotype. Furthermore, dermal lymphatic vessels in Ang2(-/-) pups prematurely recruit smooth muscle cells and do not undergo proper postnatal remodeling. In contrast, Ang2 knock-out Ang1 knock-in mice do develop a hierarchal lymphatic vasculature, suggesting that activation of Tie-2 is required for normal lymphatic development. Taken together, this work pinpoints a specific lymphatic defect of Ang2(-/-) mice and further defines the sequential steps in lymphatic vessel remodeling.

Lymphangiogenesis: Mechanisms, significance and clinical implications

Great attention has been directed toward understanding angiogenesis over the past several decades since this phenomenon was reproduced in vitro in endothelial cell and mixed vascular tissue cultures (Folkman and Hauden-schild, 1980; Folkman, 1995). The focus, however, has been almost entirely on blood vessel growth, or what we have termed “hemangiogenesis” (M. Witte and Witte, 1987 c). Yet a vast interstitial fluid circulation suffuses the tissues, bathes the parenchymal cells and interconnects with the blood vasculature via the lymphatic vasculature (Fig. 1). This arc of the circulatory system on the dark side of the blood-tissue interface and its growth (“lymphangiogenesis”) has received scant attention even though lymphatic (re)generation is both vigorous and essential, and disorders of lymphatic dynamics are common, often disfiguring, and occasionally life- and limb-threatening.

Establishment of primary cultures of human brain microvascular endothelial cells to provide an in vitro cellular model of the blood-brain barrier

We describe a method for generating primary cultures of human brain microvascular endothelial cells (HBMVECs). HBMVECs are derived from microvessels isolated from temporal tissue removed during operative treatment of epilepsy. The tissue is mechanically fragmented and size filtered using polyester meshes. The resulting microvessel fragments are placed onto type I collagen-coated flasks to allow HBMVECs to migrate and proliferate. The overall process takes less than 3 h and does not require specialized equipment or enzymatic processes. HBMVECs are typically cultured for approximately 1 month until confluent. Cultures are highly pure (∼97% endothelial cells; ∼3% pericytes), are reproducible, and show characteristic brain endothelial markers (von Willebrand factor, glucose transporter-1) and robust expression of tight and adherens junction proteins as well as caveolin-1 and efflux protein P-glycoprotein. Monolayers of HBMVECs show characteristically high transendothelial electric resistance and have proven useful in multiple functional studies for in vitro modeling of the human blood-brain barrier.

Alzheimer disease macrophages shuttle amyloid-beta from neurons to vessels, contributing to amyloid angiopathy

Neuronal accumulation of oligomeric amyloid-β (Αβ) is considered the proximal cause of neuronal demise in Alzheimer disease (AD) patients. Blood-borne macrophages might reduce Aβ stress to neurons by immigration into the brain and phagocytosis of Αβ. We tested migration and export across a blood-brain barrier model, and phagocytosis and clearance of Αβ by AD and normal subjects’ macrophages. Both AD and normal macrophages were inhibited in Αβ export across the blood-brain barrier due to adherence of Aβ-engorged macrophages to the endothelial layer. In comparison to normal subjects’ macrophages, AD macrophages ingested and cleared less Αβ, and underwent apoptosis upon exposure to soluble, protofibrillar, or fibrillar Αβ. Confocal microscopy of stained AD brain sections revealed oligomeric Aβ in neurons and apoptotic macrophages, which surrounded and infiltrated congophilic microvessels, and fibrillar Aβ in plaques and microvessel walls. After incubation with AD brain sections, normal subjects’ monocytes intruded into neurons and uploaded oligomeric Aβ. In conclusion, in patients with AD, macrophages appear to shuttle Aβ from neurons to vessels where their apoptosis may release fibrillar Aβ, contributing to cerebral amyloid angiopathy.

Angiopoietin-2 in experimental colitis

Background: The pathophysiology of inflammatory bowel disease (IBD) includes leukocyte infiltration, blood and lymphatic remodeling, weight loss and protein enteropathy. The roles of angiopoietin‐2 (Ang‐2) in initiating gut inflammation, leukocyte infiltration and angiogenesis are not well understood. Methods: Disease activity index, histopathological scoring, myeloperoxidase assay, immunohistochemistry and sodium dodecyl sulphate‐ polyacrylamide gel electrophoretic methods were employed in the present study to addess the roles of Ang‐2 in experimental colitis. Results: Several important differences were seen in the development of experimental IBD in Ang‐2−/− mice. Although weight change and disease activity differ only slightly in WT and Ang‐2−/− + DSS treated mice, leukocyte infiltration, inflammation and blood and lymphatic vessel density is significantly attenuated compared to WT + DSS mice. Gut capillary fragility and water export (stool blood and form) appear significantly earlier in Ang‐2−/− + DSS mice vs. WT. Colon lengths were also significantly reduced in Ang‐2−/− and gut histopathology was less severe in Ang‐2−/− compared to WT + DSS. Lastly, the decrease in serum protein content in WT + DSS was less severe in Ang‐2−/− + DSS, thus protein losing enteropathy (PLE) a feature of IBD is relieved by Ang‐2−/−. Conclusion: These data demonstrate that in DSS colitis, Ang‐2 mediates inflammatory hemangiogenesis, lymphangiogenesis and neutrophil infiltration to reduce some, but not all clinical features of IBD. The implications for Ang‐2 manipulation in the development of IBD and other inflammatory diseases and treatments involving Ang‐2 are discussed. (Inflamm Bowel Dis 2009)

Abnormal recruitment of periendothelial cells to lymphatic capillaries in digestive organs of angiopoietin-2-deficient mice.

The fine structure of lymphatic capillaries in the digestive organs of angiopoietin-2 (Ang2) knockout mice was studied by using both immunohistochemistry and electron microscopy. The genetic deletion of Ang2 yielded hypoplasia and disorganization of the lymphatic capillaries, with their shapes being irregular, and an aberrant recruitment of vascular periendothelial cells immunopositive for smooth muscle actin to the lymphatic capillaries. The abnormal lymphatic periendothelial cells were considered to be a type of pericyte for the lymphatic capillaries after the deletion of Ang2, because they were ultrastructurally characterized by abundant thin myofilaments in their cytoplasm and long cytoplasmic extensions similar to those shown by blood vascular pericytes. The genetic replacement of Ang2 with Ang1 rescued the defects, viz., the disorganization and disordered structure of the lymphatic capillaries. The present findings suggest that Ang2 serves the morphogenesis of lymphatic capillaries as an agonist for the receptor, Tie2, and that Ang1 can replace Ang2 in guiding lymphatic formation and development.

Inhibition of normal and experimental angiotumor endothelial cell proliferation and cell cycle progression by 2-methoxyestradiol.

Abstract With rapid growth and metabolism, aggressive cancers require an extensive vascular network, termed tumor angiogenesis. The body produces a variety of natural angiogenic inhibitors, among which is the mammalian estrogen metabolite, 2-methoxyestradiol (2-MeOE2). In this study, we compared the effects of 2-MeOE2 on a human umbilical vein cell line (HUVEC-C) and on an immortal, angiotumor-producing rat sinusoidal endothelial cell line (RSE-1). In vitro, the effects of varying concentrations of 2-MeOE2 from 0.01-100.0 μM were measured with cell counts and compared to control cells. HUVEC-C had an ED50 ~3.5 μM with ~27% inhibition of cell growth whereas RSE-1 had an ED50 ~2.2 μM with ~50% inhibition of cell growth compared with controls. The lowest concentration with maximal effect was 10.0 μM2-MeOE2 for both cell lines. Using this concentration, flow cytometric analysis of cell cycles was performed with propidium iodide stained DNA of HUVEC-C and RSE-1 at 24 and 48 hr. Both demonstrated a significant (P < 0.0001) block at G2M of the cell cycle. At 48 hr, HUVEC-C had 32% of cells in G2M (control = 9% G2M), and RSE-1 had 36% of cells in G2M (control = 18% G2M). These findings demonstrate a strong in vitro antiproliferative effect of 2-MeOE2 on normal dividing endothelial as well as angiotumor cells mediated through a cell cycle-specific block at G2M. The antiendothelial, antiangiotumor effect of 2-MeOE2 supports its potential as a therapeutic agent against solid organ cancers, benign or malignant vascular growths, and other pathologic states dependent on angiogenesis.

Chy-3 mice are Vegfc haploinsufficient and exhibit defective dermal superficial to deep lymphatic transition and dermal lymphatic hypoplasia

Recent advances in molecular lymphology and lymphatic phenotyping techniques in small animals offer new opportunities to delineate mutant mouse models. Chy‐3 mutant mice were originally named for their chylous ascites, but the underlying lymphatic disorder was not defined. We now re‐examined these mice and applied advanced genotyping and lymphatic phenotyping techniques to pinpoint the specific lymphatic defect in this mouse model. We demonstrated that Chy‐3 mice carry a large chromosomal deletion that includes Vegfc and narrowed this region by monitoring the heterozygosity of genetic markers. We found that Chy‐3 mice not only exhibited chylous ascites but also lymphedema of the hind paws and, in approximately half of the males, lymphedema of the penis. Visual lymphangiography and immunofluorescence staining showed a hypoplastic dermal lymphatic network, whereas the blood vasculature appeared unaffected. This hypoplastic lymphatic network was functional, and all adult Chy‐3 mice exhibited a lateral lymphatic pathway directly connecting the inguinal to the axillary lymph node. The dermal superficial to deep lymphatic connections in upper limbs and in all cervical regions were intact and functionally drained the upper body. Lymphatic tracer was not transported from the dermal to the deep truncal lymphatic system in the lower limbs, even though the deep lymphatic vessels and nodes were present and patent. These findings further delineate the lymphatic phenotype of Chy‐3 mice, identify a collateral lymph drainage pathway previously undescribed in other genetic models of lymphedema, and demonstrate a predilection for lymphatic abnormalities of the lower limbs. Developmental Dynamics 236:2346–2355, 2007.

Segregation analyses and a genome‐wide linkage search confirm genetic heterogeneity and suggest oligogenic inheritance in some Milroy congenital primary lymphedema families

We previously described six families with Milroy congenital lymphedema, only one of which showed possible linkage to a candidate locus on chromosome 5 [Witte et al., 1998]. We have now performed a complex segregation analysis of these families, and performed linkage analyses with the other 387 markers used in our genome-wide search. Our results confirm that Milroy lymphedema is generally inherited as a dominant condition. However, this mode of inheritance, as elucidated from the segregation analyses, did not account for all observed familial correlations. The segregation analysis also suggested that shared environmental or additional genetic factors are important in explaining the observed familial aggregation. The finding of linkage to multiple locations in the largest family studied by multipoint parametric mapping (one of which was confirmed by sib-pair non-parametric mapping), suggests that Milroy congenital lymphedema may be oligogenic in this family.

Elevated Levels of Bilirubin and Long-Term Exposure Impair Human Brain Microvascular Endothelial Cell Integrity

The pathogenesis of encephalopathy by unconjugated bilirubin (UCB) seems to involve the passage of high levels of the pigment across the blood-brain barrier (BBB) and the consequent damage of neuronal cells. However, it remains to be clarified if and how the disruption of BBB occurs by UCB. We used confluent monolayers of human brain microvascular endothelial cells (HBMEC) to explore the sequence of events produced by UCB. A cell line and primary cultures of HBMEC were exposed to 50 or 100 µM UCB, in the presence of 100 µM human serum albumin, to mimic moderate and severe jaundice, for 1-72 h. UCB caused loss of cell viability in a concentration-dependent manner. UCB inhibited the secretion of interleukin-6, interleukin-8, monocyte chemoattractant protein-1 and vascular endothelial growth factor at early time points, but enhanced their secretion at later time points. Upregulation of mRNA expression, particularly by 100 µM UCB, preceded cytokine secretion. Other early events include the disruption of glutathione homeostasis and the increase in endothelial nitric oxide synthase expression followed by nitrite production. Prolonged exposure to UCB upregulated the expression of β-catenin and caveolin-1. In conclusion, elevated concentrations of UCB affect the integrity of HBMEC monolayers mediated by oxidative stress and cytokine release. UCB also induced increased expression of caveolin-1, which has been associated with BBB breakdown, and β-catenin, probably as an attempt to circumvent that impairment. These findings provide a basis for target-directed therapy against brain endothelial injury caused by UCB.