LYL1 activity is required for the maturation of newly formed blood vessels in adulthood

Abstract

The 2 related basic helix loop helix genes, LYL1 and TAL-1 are active in hematopoietic and endothelial lineages. While Tal-1 is essential for both hematopoietic and vascular development, the role of Lyl1 appears to be distinct as deficient mice are viable and display modest hematopoietic defects. Here, we reveal a role for Lyl1 as a major regulator of adult neovascularization. Tumors implanted into Lyl1-deficient mice showed higher proliferation and angiogenesis, as evidenced by enlarged lumens, reduced pericyte coverage and increased permeability, compared with wild type littermates. Of note, Lyl1-deficient tumor vessels exhibited an up-regulation of Tal-1, the VE-Cadherin target gene, as well as Angiopoietin-2, 3 major actors in angiogenesis. Hematopoietic reconstitution experiments demonstrated that this sustained tumor angiogenesis was of endothelial origin. Moreover, the angiogenic phenotype observed in the absence of Lyl1 function was not tumor-restricted as microvessels forming in Matrigel or originating from aortic explants were also more numerous and larger than their wild-type counterparts. Finally, LYL1 depletion in human endothelial cells revealed that LYL1 controls the expression of molecules involved in the stabilization of vascular structures. Together, our data show a role for LYL1 in the postnatal maturation of newly formed blood vessels. (Blood. 2010; 115(25):5270-5279) IntroductionThe vascular system through development and adulthood answers to injury and remodeling and is one of the key systems sustaining normal physiology. Conversely, its deregulation also underlies multiple pathologic processes such as ischemia, inflammation and tumor. Angiogenesis normally occurs as a sequential series of morphogenetic events resulting in a functional network of vessels. Regulation of endothelial cell function in all these processes requires the integration of complex signals by the coordinated action of specific transcription factors.LYL1 is a member of the large basic helix loop helix (bHLH) family, and is closely related to TAL-1 (also called SCL). Both LYL1 and TAL-1 were identified through their involvement in chromosomal rearrangements in human T-cell leukemia (reviewed in Baer 1 ). Tal-1 deficiency causes embryonic lethality due to the absence of hematopoietic cell formation [2][3][4][5] and several studies have shown that Tal-1 is also involved in the formation of the vascular system. 6-10 Unlike Tal-1, Lyl1 is not essential for developmental processes, since homozygous disruption of Lyl1 activity induces only a mild hematopoietic phenotype. 11 The viable Lyl1-deficient mice display a reduced number of B cells and impaired long-term hematopoietic reconstitution capacity. 11 The highly conserved bHLH motifs between TAL-1 and LYL1 are functionally interchangeable to restore immature hematopoiesis in Tal-1 deficient embryonic stem cells. 4,12 Moreover, Tal-1 and Lyl1 display redundant activities to maintain adult hematopoietic stem cell functions. 13 However, full-length LYL1 cannot rescue early letha...

Lung endothelial barrier disruption in Lyl1-deficient mice

“…We previously reported that lack of LYL1 affects the maturation of newly formed blood vessels in adult mice and reduces active Rap1 in ECs (32). Here, we completed our analysis of LYL1 function by showing that it is required also for the formation of the endothelial barrier specifically in mature lungs by acting upstream of VE-cadherin and of RhoA.…”

“…We previously reported that LYL1 silencing in human ECs downregulate the expression of several molecules that contribute to endothelial junction maturation (32), namely integrin-␣2, which has an important role in EC-ECM attachment (6), as well as RapGEF1 and RapGEF2, which have multiple roles through Rap1 activation in AJ maturation (30). In this study, we show that LYL1 also regulates the activity of RhoA in ECs and that LYL1 downregulation leads to impaired AJs.…”

“…All experiments were conducted by authorized personnel (agreement numbers: 34-308 for N. Pirot and 34-368 for V. Pinet) and approved by the Institutional Review Board at the Animal Facility of the Institut de Génétique Moléculaire de Montpellier. Animals were genotyped by PCR analysis of tail DNA as described elsewhere (32). Mice were anesthetized by isofluorane inhalation and killed at 2, 4, 6, 9, and 12 mo of age; lungs were collected and fixed in neutral buffered formalin (4% formaldehyde) overnight, dehydrated, and embedded in paraffin.…”

“…Primary ECs from human umbilical vein (HUVECs; PromoCell) and the immortalized human EC line human telomerase reverse transcriptase (hTERT-1) (51) were obtained from the United States Center for Disease Control and were cultured in complete endothelial growth medium-2 (PromoCell) supplemented with 3% fetal calf serum (FCS) (HyClone, Perbio Science). Small-interfering RNA (siRNA) transfection in HUVECs and small-hairpin RNA (shRNA) transduction in hTERT-1 cells were performed as previously described (32). The sequences of duplex RNAs were as follows: LYL1A-Forward: GAUGGAGCAAACCGCUUUG-TT; LYL1A-Reverse: CAAAGCGGUUUGCUCCAUC-TT; LYL1D-Forward: GU-CAAGGAAAGGGCAGUGG-TT; LYL1D-Reverse: CCACUGC-CCUUUCCUUGAC-TT.…”

“…We recently identified angiopoietin-2, a major regulator of angiogenesis and lymphangiogenesis (5), as the first direct LYL1 target gene in human ECs (15). In adult mice, Lyl1 is expressed in both quiescent and angiogenic vessels and is required for the late stages of postnatal angiogenesis to limit the growth of newly formed vascular structures (32). Indeed, syngeneic tumors implanted in Lyl1Ϫ/Ϫ mice develop vessels with enlarged lumens, reduced pericyte coverage, and increased permeability.…”