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. 2010 Feb 25;115(8):1654-61.
doi: 10.1182/blood-2009-07-235655. Epub 2009 Nov 10.

ALK1 signaling regulates early postnatal lymphatic vessel development

Kyle Niessen  1 , Gu Zhang, John Brady Ridgway, Hao Chen, Minhong Yan
Affiliations

ALK1 signaling regulates early postnatal lymphatic vessel development

Kyle Niessen et al. Blood. .

Abstract

In vertebrates, endothelial cells form 2 hierarchical tubular networks, the blood vessels and the lymphatic vessels. Despite the difference in their structure and function and genetic programs that dictate their morphogenesis, common signaling pathways have been recognized that regulate both vascular systems. ALK1 is a member of the transforming growth factor-beta type I family of receptors, and compelling genetic evidence suggests its essential role in regulating blood vascular development. Here we report that ALK1 signaling is intimately involved in lymphatic development. Lymphatic endothelial cells express key components of the ALK1 pathway and respond robustly to ALK1 ligand stimulation in vitro. Blockade of ALK1 signaling results in defective lymphatic development in multiple organs of neonatal mice. We find that ALK1 signaling regulates the differentiation of lymphatic endothelial cells to influence the lymphatic vascular development and remodeling. Furthermore, simultaneous inhibition of ALK1 pathway increases apoptosis in lymphatic vessels caused by blockade of VEGFR3 signaling. Thus, our study reveals a novel aspect of ALK1 signaling in regulating lymphatic development and suggests that targeting ALK1 pathway might provide additional control of lymphangiogenesis in human diseases.

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Figures

Figure 1
Figure 1
Lymphatic endothelial cells express ALK1. (A) RT-PCR analysis of gene expression in HUVECs and human microvascular dermal neonatal lymphatic endothelial cells. RNA from a mixture of human cell lines with (+) and without (−) reverse transcriptase enzyme was used as a positive and negative control. Sizes of PCR products are indicated. (B) Expression level of Smad6 in HUVECs and lymphatic endothelial cells stimulated with 50 pg/mL rhBMP9 or 500 pg/mL rhBMP10 for 3 hours. Inhibition of rhBMP9 and rhBMP10 induced up-regulation of Smad6 expression by pretreatment with 10 μg/mL ALK1Fc. Quantitative RT-PCR results are normalized to glyceraldehyde 3-phosphate dehydrogenase and then to the untreated (UT) sample. Gray squares represent individual data points (n = 3). (C) Expression level of Smad6 in lymphatic endothelial cells stimulated with rhBMP9 150 pg/mL and transfected with siRNA targeting the Alk1-7, Smad1, 4, 5, or endoglin (Eng). Gray squares represent individual data points (n = 3).
Figure 2
Figure 2
ALK1Fc causes vascular and lymphatic defects. (A) Isolectin-B4 (green) and SMA (red) staining of P8 retina from PBS- or ALK1Fc-treated pups (10 mg/kg; P1 P3, P5). (B) Analysis of lymphatic function by dextran–FITC injection into the tail tip () of a P5 pup treated with PBS or ALK1Fc (10 mg/kg, P1 and P3). Images were captured 5 minutes after injection. (Top) Drainage of dextran–FITC into the collecting lymphatic vessel that extends the length of the tail. Scale bar represents 1 mm. (Bottom) “Honeycomb” lymphatic vessels in the dermis adjacent to the injection site (box). Scale bar represents 250 μm. (C) CD31 (blue, vasculature) and LYVE1 (red, lymphatic) staining of the tail dermis from PBS- or ALK1Fc-treated pups (10 mg/kg, P1 and P3) at P6. Scale bar represents 250 μm. (D) CD31 (blue, vasculature) and LYVE1 (red, lymphatic) staining of the intestine from PBS- or ALK1Fc-treated pups (10 mg/kg, P1 and P3) at P6. Scale bar represents 250 μm.
Figure 3
Figure 3
The ALK1 pathway is directly involved in lymphatic vessel development. (A) Analysis of lymphatic and vascular development at P6, after treatment with ALK1Fc, ACVR2BFc, BMPR2Fc, or VEGFR3Fc (10 mg/kg, P1 and P3). Lymphatic development is visualized by LYVE1 (green) staining in the tail dermis. Vascular development is visualized by isolectin-B4 (green) and SMA (red) staining of the retina. Scale bar represents 250 μm. (B) Inhibition of rhBMP9 induced up-regulation of Smad6 expression by the ALK1 immunized serum (α-ALK1-s) or affinity purified antibody (α-ALK1-a), whereas preimmune serum (Pre) has no effect. Red boxes represent each data point (n = 3). (C) Inhibition of rhBMP9 binding to ALK1 protein by treatment with the ALK1 immunized serum (α-ALK1-s) or affinity purified antibody (α-ALK1-a), whereas preimmune serum (Pre) has no effect. Red boxes represent each data point (n = 2). (D) ALK1-neutralizing antibody binds mouse ALK1 but not ALK2, 3, 4, 5, 6, or 7. (E) Analysis of lymphatic development at P6 after treatment with the ALK1 neutralizing antibody (10 mg/kg, P1 and P3) in the tail dermis. (F) Analysis of lymphatic development at P6 after treatment with the ALK1-neutralizing antibody (10 mg/kg, P1 and P3) in the ear. Lymphatic development is visualized by LYVE1 (green) staining in the tail dermis and ear. Scale bar represents 250 μm.
Figure 4
Figure 4
ALK1 is involved in multiple stages of lymphatic development. (A) Analysis of vascular and lymphatic development in P8 pups when ALK1Fc treatment is started when the honeycomb pattern is being established (10 mg/kg, P3 and P5). Lymphatic development is visualized by LYVE1 (green) staining in the tail dermis, whereas vascular development is visualized by isolectin-B4 (green) staining of the retina. Scale bar represents 500 μm (retina) and 250 μm (tail). (B) Analysis of lymphatic development in P12 pups when ALK1 treatment is started when the honeycomb structure is fully established (10 mg/kg, P8 and P10). Lymphatic development is visualized by LYVE1 (green) staining in the tail dermis. Scale bar represents 250 μm. (C) High-magnification images demonstrate the remodeling process that occurs from P8 to P12 and the “ringed” structures that fail to be remodeled in ALK1Fc-treated pups.
Figure 5
Figure 5
Distinct roles of ALK1 and VEGFR3 during lymphatic development. (A top) Analysis of lymphatic development in P4 pups after treatment with ALK1Fc (10 mg/kg, P2), VEGFR3Fc (1 mg/kg, P1), or combined ALK1Fc and VEGFR3Fc. Lymphatic development is visualized by LYVE1 staining (red), inset shows a greater magnification image of the honeycomb structure that is disrupted by the treatments. Scale bar represents 500 μm. (Middle) Analysis of podoplanin (green) and LYVE1 (red) expression after ALK1Fc, VEGFR3Fc, or combination treatments. Scale bar represents 250 μm. (Bottom) Analysis of LYVE1 (green) and active Caspase3 (red) expression after ALK1Fc, VEGFR3Fc, and combination treatment. Scale bar represents 250 μm. (B) Analysis of lymphatic development in P8 pups after ALK1Fc (10 mg/kg, P3 and P5), VEGFR3Fc (10 mg/kg, P6 or P7), or combined ALK1Fc and VEGFR3Fc treatment. Lymphatic development is visualized by LYVE1 staining (red). Scale bar represents 250 μm.
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Comment in

  • Jazzing up vessel growth.
    Kahn ML. Kahn ML. Blood. 2010 Feb 25;115(8):1479. doi: 10.1182/blood-2009-11-254995. Blood. 2010. PMID: 20185593 No abstract available.

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