Ascending Vasa Recta Are Angiopoietin/Tie2-Dependent Lymphatic-Like Vessels.

Urinary concentrating ability is central to mammalian water balance and depends on a medullary osmotic gradient generated by a countercurrent multiplication mechanism. Medullary hyperosmolarity is protected from washout by countercurrent exchange and efficient removal of interstitial fluid resorbed from the loop of Henle and collecting ducts. In most tissues, lymphatic vessels drain excess interstitial fluid back to the venous circulation. However, the renal medulla is devoid of classic lymphatics. Studies have suggested that the fenestrated ascending vasa recta (AVRs) drain the interstitial fluid in this location, but this function has not been conclusively shown. We report that late gestational deletion of the angiopoietin receptor endothelial tyrosine kinase 2 (Tie2) or both angiopoietin-1 and angiopoietin-2 prevents AVR formation in mice. The absence of AVR associated with rapid accumulation of fluid and cysts in the medullary interstitium, loss of medullary vascular bundles, and decreased urine concentrating ability. In transgenic reporter mice with normal angiopoietin-Tie2 signaling, medullary AVR exhibited an unusual hybrid endothelial phenotype, expressing lymphatic markers (prospero homeobox protein 1 and vascular endothelial growth factor receptor 3) as well as blood endothelial markers (CD34, endomucin, platelet endothelial cell adhesion molecule 1, and plasmalemmal vesicle-associated protein). Taken together, our data redefine the AVRs as Tie2 signaling-dependent specialized hybrid vessels and provide genetic evidence of the critical role of AVR in the countercurrent exchange mechanism and the structural integrity of the renal medulla.

G-CSF rescues tumor growth and neo-angiogenesis during liver metastasis under host angiopoietin-2 deficiency.

Suppression of neo-angiogenesis is a clinically used anti-tumor strategy with new targets such as angiopoietin-2 (Ang2) being proposed. However, the functions of Ang2 in vascular remodeling, inflammation and tumor growth are not consistent. We examined effect of depletion of host Ang2 on liver colony formation using Ang2 deficient (Ang2(-/-)) mice. Surprisingly, the metastatic colonies formed in Ang2(-/-) mice were larger than those in the wild type. These colonies had greater vascular density with more pericyte coverage than the vessels in liver colonies in the wild type. Liver VEGF concentration in both genotypes was equivalent, and thus, the differences appeared VEGF independent. However, after colony formation, the serum concentration of granulocyte-colony stimulating factor (G-CSF) and CXCL1 in Ang2(-/-) mice was 12 and 6 times greater than after colony formation in wild type. Increase of these two cytokines was associated with two times greater numbers of neutrophils recruited to the liver. Two times more Tie2+/CD11b+/CD31- cells were present in the tumors in Ang2(-/-) than in the wild type livers. These results suggest that the depletion of host Ang2 induced compensatory VEGF-independent angiogenic mechanisms and thus enhanced liver metastatic colony growth and colony vascularity. They further indicate organotypic differences in response to tumor metastasis. In contrast, Ang2 deficiency inhibited tumor growth during metastatic colony formation in the lung, consistent with the reports of decreased pulmonary seeding of tumor cells after pharmacological inhibition of Ang2. Further studies are thus required to assess the effects of pharmacological Ang2 blockade for cancer patients particularly in the liver.

Decreased hypoxia-induced neovascularization in angiopoietin-2 heterozygous knockout mouse through reduced MMP activity.

BACKGROUND/AIMS: Proliferative diabetic retinopathy is characterized by the formation of retinal neovascularization. Angiopoietin-2 (Ang-2) and matrix metalloproteinase (MMP) play a critical role in angiogenesis. However, the precise location and function of Ang-2 during formation of retinal neovascularizations driven by hypoxia in relation to MMP activity have not been elucidated. In this study, we investigated the response of Ang-2 heterozygous knockout retinas (Ang2(+/-) mouse) to hypoxia and its link to MMP activity in an oxygen-induced retinopathy (OIR) model. METHODS: Pre-retinal neovascularizations were quantitated in vertical sections. Intra-retinal angiogenesis was assessed by whole mount immunofluorescence staining of retinas. MMP activity was examined in retinal protein lysate and whole mount retinal in situ zymography. RESULTS: Ang2(+/-) retinas subjected to the OIR model showed 33% reduced neovascularization and 271% increased avascular zones at postnatal day 17. In the OIR model, Ang-2 was modestly expressed in pre-retinal neovascularizations and venules, but strongly in arterioles and capillary sprouts. MMPs were activated in close association to where Ang-2 is expressed. MMP activity was substantially decreased in Ang2(+/-) retinas. CONCLUSIONS: Our present data suggest the spatially concomitant expression of Ang2 and MMPs, and that Ang2 modulates hypoxia-induced neovascularization by regulating MMP activity.

Host-derived angiopoietin-2 affects early stages of tumor development and vessel maturation but is dispensable for later stages of tumor growth.

The angiopoietin/Tie2 system has been identified as the second vascular-specific receptor tyrosine kinase system controlling vessel assembly, maturation, and quiescence. Angiopoietin-2 (Ang-2) is prominently up-regulated in the host-derived vasculature of most tumors, making it an attractive candidate for antiangiogenic intervention. Yet, the net outcome of Ang-2 functions on tumor angiogenesis is believed to be contextual depending on the local cytokine milieu. Correspondingly, Ang-2 manipulatory therapies have been shown to exert protumorigenic as well as antitumorigenic effects. To clarify the role of Ang-2 for angiogenesis and tumor growth in a definite genetic experimental setting, the present study was aimed at comparatively studying the growth of different tumors in wild-type and Ang-2-deficient mice. Lewis lung carcinomas, MT-ret melanomas, and B16F10 melanomas all grew slower in Ang-2-deficient mice. Yet, tumor growth in wild-type and Ang-2-deficient mice dissociated during early stages of tumor development, whereas tumor growth rates during later stages of primary tumor progression were similar. Analysis of the intratumoral vascular architecture revealed no major differences in microvessel density and perfusion characteristics. However, diameters of intratumoral microvessels were smaller in tumors grown in Ang-2-deficient mice, and the vasculature had an altered pattern of pericyte recruitment and maturation. Ang-2-deficient tumor vessels had higher pericyte coverage indices. Recruited pericytes were desmin and NG2 positive and predominately alpha-smooth muscle actin negative, indicative of a more mature pericyte phenotype. Collectively, the experiments define the role of Ang-2 during tumor angiogenesis and establish a better rationale for combination therapies involving Ang-2 manipulatory therapies.

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.

Angiopoietin-2 deficiency decelerates age-dependent vascular changes in the mouse retina.

Retinae of aged humans show signs of vascular regression. Vascular regression involves a mismatch between Angiopoietin-2 (Ang-2) and vascular endothelial growth factor (VEGF) expression. We used heterozygous Ang-2 deficient (Ang2LacZ) mice to evaluate murine retinal vascular changes and gene expression of growth factors. Vascular changes were assessed by quantitative retinal morphometry and gene expression levels of growth factors were measured by quantitative PCR. The numbers of endothelial cells and pericytes did not change in the Ang2LacZ retinae with age, whereas they decreased throughout the age spectrum studied in the wild type retinae. Moreover, vascular regression significantly decelerated in the heterozygous Ang2LacZ retinae (200% to 1 month), while the formation of acellular capillaries was significantly increased at 13 months in the wild type retinae (340% to 1 month). Gene expression analysis revealed that VEGF, Ang-1, PDGF-B and Ang2 mRNA levels were decreased in the wild type retinae at 9 month of age. However, the decrease of Ang-2 was smaller compared with other genes. While VEGF levels dropped in wild type mice up to 60% compared to 1 month, VEGF increased in heterozygous Ang-2 deficient retinae at an age of 9 months (141% to 1 month). Similarly, Ang-1 levels decreased in wild type mice (45% to 1 month), but remained stable in Ang2LacZ mice. These data suggest that Ang-2 gene dose reduction decelerates vasoregression in the retina with age. This effect links to higher levels of survival factors such as VEGF and Ang-1, suggesting that the ratio of these factors is critical for capillary cell survival.

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.

Angiopoietin-2 sensitizes endothelial cells to TNF-alpha and has a crucial role in the induction of inflammation.

The angiopoietins Ang-1 and Ang-2 have been identified as ligands of the receptor tyrosine kinase Tie-2 (refs. 1,2). Paracrine Ang-1-mediated activation of Tie-2 acts as a regulator of vessel maturation and vascular quiescence. In turn, the antagonistic ligand Ang-2 acts by an autocrine mechanism and is stored in endothelial Weibel-Palade bodies from where it can be rapidly released upon stimulation. The rapid release of Ang-2 implies functions of the angiopoietin-Tie system beyond its established role during vascular morphogenesis as a regulator of rapid vascular responses. Here we show that mice deficient in Ang-2 (encoded by the gene Angpt2) cannot elicit an inflammatory response in thioglycollate-induced or Staphylococcus aureus-induced peritonitis, or in the dorsal skinfold chamber model. Recombinant Ang-2 restores the inflammation defect in Angpt2(-/-) mice. Intravital microscopy showed normal TNF-alpha-induced leukocyte rolling in the vasculature of Angpt2(-/-)mice, but rolling cells did not firmly adhere to activated endothelium. Cellular experiments showed that Ang-2 promotes adhesion by sensitizing endothelial cells toward TNF-alpha and modulating TNF-alpha-induced expression of endothelial cell adhesion molecules. Together, these findings identify Ang-2 as an autocrine regulator of endothelial cell inflammatory responses. Ang-2 thereby acts as a switch of vascular responsiveness exerting a permissive role for the activities of proinflammatory cytokines.

Dysmorphogenesis of kidney cortical peritubular capillaries in angiopoietin-2-deficient mice.

Angiopoietin-2 (Ang-2) modulates Tie-2 receptor activation. In mouse kidney maturation, Ang-2 is expressed in arteries, with lower levels in tubules, whereas Tie-2 is expressed by endothelia. We hypothesized that Ang-2 deficiency disrupts kidney vessel patterning. The normal renal cortical peritubular space contains fenestrated capillaries, which have few pericytes; they receive water and solutes which proximal tubules reclaim from the glomerular filtrate. In wild-type neonates, alpha smooth muscle actin (alpha SMA), platelet-derived growth factor receptor beta (PDGFR beta), and desmin-expressing cells were not prominent in this compartment. In Ang-2 null mutants, alpha SMA, desmin, and PDGFR beta prominently immunolocalized in cortical peritubular locations. Some alpha SMA-positive cells were closely associated with CD31- and Tie-2-positive peritubular capillary endothelia, and some of the alpha SMA-positive cells expressed PDGFR beta, desmin, and neural/glial cell 2 (NG2), consistent with a pericyte-like identity. Immunoblotting suggested an increase of total and tyrosine-phosphorylated Tie-2 proteins in null mutant versus wild-type kidneys, and electron microscopy confirmed disorganized capillaries and adjacent cells in cortical peritubular spaces in mutant neonate kidneys. Hence, Ang-2 deficiency causes dysmorphogenesis of cortical peritubular capillaries, with adjacent cells expressing pericyte-like markers; we speculate the latter effect is caused by disturbed paracrine signaling between endothelial and surrounding mesenchymal precursor cells.

Angiopoietin-2 causes pericyte dropout in the normal retina: evidence for involvement in diabetic retinopathy.

Pericyte loss is an early pathologic feature of diabetic retinopathy, consistently present in retinae of diabetic humans and animals. Because pericyte recruitment and endothelial cell survival are controlled, in part, by the angiopoietin/Tie2 ligand/receptor system, we studied the expression of angiopoietin-2 and -1 in relation to the evolution of pericyte loss in diabetic rat retinae, using quantitative retinal morphometry, and in retinae from mice with heterozygous angiopoietin deficiency (Ang-2 LacZ knock-in mice). Finally, recombinant angiopoietin-2 was injected into eyes of nondiabetic rats, and pericyte numbers were quantitated in retinal capillaries. Angiopoietin-1 protein was present in the normal maturing retina and was upregulated 2.5-fold in diabetic retinae over 3 months of diabetes. In contrast, angiopoietin-2 protein was consistently upregulated more than 30-fold in the retinae of diabetic rats, preceding the onset of pericyte loss. Heterozygous angiopoietin-2 deficiency completely prevented diabetes-induced pericyte loss and reduced the number of acellular capillary segments. Injection of angiopoietin-2 into the eyes of normal rats induced a dose-dependent pericyte loss. These data show that upregulation of angiopoietin-2 plays a critical role in the loss of pericytes in the diabetic retina.