Inductive angiocrine signals from sinusoidal endothelium are required for liver regeneration

Abstract

During embryogenesis, endothelial cells induce organogenesis before the development of circulation. These findings suggest that endothelial cells not only form passive conduits to deliver nutrients and oxygen, but also establish an instructive vascular niche, which through elaboration of paracrine trophogens stimulates organ regeneration, in a manner similar to endothelial-cell-derived angiocrine factors that support haematopoiesis. However, the precise mechanism by which tissue-specific subsets of endothelial cells promote organogenesis in adults is unknown. Here we demonstrate that liver sinusoidal endothelial cells (LSECs) constitute a unique population of phenotypically and functionally defined VEGFR3(+)CD34(-)VEGFR2(+)VE-cadherin(+)FactorVIII(+)CD45(-) endothelial cells, which through the release of angiocrine trophogens initiate and sustain liver regeneration induced by 70% partial hepatectomy. After partial hepatectomy, residual liver vasculature remains intact without experiencing hypoxia or structural damage, which allows study of physiological liver regeneration. Using this model, we show that inducible genetic ablation of vascular endothelial growth factor (VEGF)-A receptor-2 (VEGFR2) in the LSECs impairs the initial burst of hepatocyte proliferation (days 1-3 after partial hepatectomy) and subsequent reconstitution of the hepatovascular mass (days 4-8 after partial hepatectomy) by inhibiting upregulation of the endothelial-cell-specific transcription factor Id1. Accordingly, Id1-deficient mice also manifest defects throughout liver regeneration, owing to diminished expression of LSEC-derived angiocrine factors, including hepatocyte growth factor (HGF) and Wnt2. Notably, in in vitro co-cultures, VEGFR2-Id1 activation in LSECs stimulates hepatocyte proliferation. Indeed, intrasplenic transplantation of Id1(+/+) or Id1(-/-) LSECs transduced with Wnt2 and HGF (Id1(-/-)Wnt2(+)HGF(+) LSECs) re-establishes an inductive vascular niche in the liver sinusoids of the Id1(-/-) mice, initiating and restoring hepatovascular regeneration. Therefore, in the early phases of physiological liver regeneration, VEGFR2-Id1-mediated inductive angiogenesis in LSECs through release of angiocrine factors Wnt2 and HGF provokes hepatic proliferation. Subsequently, VEGFR2-Id1-dependent proliferative angiogenesis reconstitutes liver mass. Therapeutic co-transplantation of inductive VEGFR2(+)Id1(+)Wnt2(+)HGF(+) LSECs with hepatocytes provides an effective strategy to achieve durable liver regeneration.

Figure 1. Phenotypic signature and contribution of LSECs to physiological liver regeneration induced by 70% partial hepatectomy (PH)

a) Liver sections obtained from VEGFR2-GFP reporter mice. During liver regeneration VEGFR2 is exclusively expressed on the liver ECs. b) Restricted expression of VEGFR3 on LSECs, but not CD34⁺ large vessels or hepatocytes. c) Polyvariate flow cytometric analysis of the liver nonparenchymal cells. VEGFR2⁺ cells that are CD45⁻, express EC-specific VE-cadherin. d) Specific expression of VEGFR3 on VEGFR2⁺VE-cadherin⁺CD45⁻ LSECs, with a predominant fraction being CD34⁻FactorVIII⁺Prox-1⁻. Thus, LSECs could be identified as VEGFR3⁺CD34⁻ cells. e) 48 hours after PH, E-cadherin⁺P-H3⁺ mitotic hepatocytes are localized adjacent to VE-cadherin⁺ and VEGFR2⁺ ECs. f, g) Kinetics of LSECs expansion (f) and hepatocyte mitosis (g) during liver regeneration (n = 4). Hpf, high power field. Scale bars, 50 μm. Error bars, s.e.m.

Figure 2. VEGFR2-Id1 activation in LSECs mediates PH-induced liver regeneration

a, b) Hepatocyte proliferation after PH is impaired in VEGFR2^fl/fl mice (n = 5). c-e) Inhibition of liver mass regeneration (c) and functional VE-cadherin⁺isolectin⁺ vessel formation (d, e) in VEGFR2^fl/fl mice after PH (n = 4-6). f, g) Injection of VEGF-A₁₆₄, but not VEGFR1-specific ligand PlGF, accelerates the regeneration of liver mass (f), associated with an incremental increase in VEGFR3⁺CD34⁻ LSEC number (g) (n = 4). h) Regenerative liver section of Id1^VenusYFP mouse. Id1 is selectively upregulated by PH in VE-cadherin⁺ vessels. i) VEGFR2 deletion diminishes Id1 upregulation in the regenerative liver (n = 5). *P < 0.05; #P < 0.01, versus VEGFR2^fl/+ (b-e, i), versus PlGF-treated group (f). Scale bar, 50 μm. Error bars, s.e.m.

Figure 3. Id1 upregulation in LSECs is essential for liver regeneration

a) Compared to their wild type littermates (WT), Id1^−/− mice manifest impaired regeneration in liver mass, which fails to be rescued by VEGF-A₁₆₄ administration (n = 5). b, c) Impaired hepatocyte proliferation (b) and assembly of VE-cadherin⁺isolectin⁺ vessels (c) in the Id1^−/− mice after PH (n = 5). d, e) The LSEC-dependent stimulation of hepatocyte proliferation was specifically inhibited by Id1 gene knockdown. Scr, scrambled. CM, LSEC-conditioned medium (n = 4). f) Intrasplenic transplantation of GFP-marked LSECs incorporates into the lumen of VEGFR3⁺ sinusoidal vasculature in the Id1^−/− liver. g, h) Transplantation of Id1^+/+ LSECs restores the regeneration of mass (g) and hepatocyte proliferation (h) in the Id1^−/− liver (n = 4). Dashed line, level of Id1^−/− liver without EC transplantation. k) Cellular proximity is essential in the stimulation of hepatocyte mitosis by the transplanted GFP⁺Id1^+/+ vasculature. *P < 0.05, versus Id1^−/− (a); #P < 0.01, versus Id1^−/− with VEGF₁₆₄ (a), versus WT (b, c). Scale bars, 50 (d, f) and 20 (h) μm. Error bars, s.e.m.

Figure 4. Id1-mediated induction of Wnt2 and HGF in LSECs stimulates hepatic regeneration

a) Upregulation of HGF and Wnt2 is impaired in Id1^−/− LSECs after PH (n = 5). b) Intrasplenic transplantation of GFP-marked Id1^−/− LSECs carrying both Wnt2 and HGF (Id1^−/−Wnt2⁺HGF⁺GFP⁺) rescues the regeneration of Id1^−/− liver mass (n = 4). c) Transplantation of Id1^−/−Wnt2⁺HGF⁺ LSECs restores the impaired hepatocyte proliferation in the Id1^−/− liver. (n = 4). d) The proximity between the mitotic hepatocytes and the Id1^−/−Wnt2⁺HGF⁺GFP⁺ LSECs in the Id1^−/− liver. e) Requirement for VEGFR2-Id1 pathway in LSEC-mediated liver regeneration. Intrasplenic transplantation of Id1^+/+ LSECs into the Id1^−/− liver sinusoids restores hepatic-vascular regeneration. Transplanted Id1^+/+ or Id1^−/−Wnt2⁺HGF⁺GFP⁺ LSECs localize to the vicinity of hepatocytes, promoting inductive and proliferative angiogenesis thereby sustaining physiological liver regeneration. *P < 0.05; #P < 0.01. Scale bar, 20 μm. Error bars, s.e.m.