Connective tissue growth factor acts within both endothelial cells and beta cells to promote proliferation of developing beta cells

Abstract

Type 1 and type 2 diabetes result from an absolute or relative reduction in functional β-cell mass. One approach to replacing lost β-cell mass is transplantation of cadaveric islets; however, this approach is limited by lack of adequate donor tissue. Therefore, there is much interest in identifying factors that enhance β-cell differentiation and proliferation in vivo or in vitro. Connective tissue growth factor (CTGF) is a secreted molecule expressed in endothelial cells, pancreatic ducts, and embryonic β cells that we previously showed is required for β-cell proliferation, differentiation, and islet morphogenesis during development. The current study investigated the tissue interactions by which CTGF promotes normal pancreatic islet development. We found that loss of CTGF from either endothelial cells or β cells results in decreased embryonic β-cell proliferation, making CTGF unique as an identified β cell-derived factor that regulates embryonic β-cell proliferation. Endothelial CTGF inactivation was associated with decreased islet vascularity, highlighting the proposed role of endothelial cells in β-cell proliferation. Furthermore, CTGF overexpression in β cells during embryogenesis using an inducible transgenic system increased islet mass at birth by promoting proliferation of immature β cells, in the absence of changes in islet vascularity. Together, these findings demonstrate that CTGF acts in an autocrine manner during pancreas development and suggest that CTGF has the potential to enhance expansion of immature β cells in directed differentiation or regeneration protocols.

Fig. 1.

CTGF from multiple sources is required for β-cell proliferation during embryogenesis. Quantification of β-cell proliferation normalized to control littermates in CTGF^{e2COIN/e2COIN};Tie-1-Cre (A), CTGF^{e2COIN/e2COIN};Pdx-Cre (B), and CTGF^{e2COIN/e2COIN};Ngn3-Cre^BAC (C) embryos at E18.5 (A and B) and P1 (C). *P < 0.05 compared with Control. (D and E) Representative images showing decreased β-cell proliferation in CTGF^{e2COIN/e2COIN};Tie-1-Cre embryos (E) compared with controls (D). Arrowheads indicate proliferating β cells. n = 3–5 animals per genotype. (Magnification: 400×.)

Fig. 2.

Different sources of CTGF function redundantly to promote lineage allocation and islet morphogenesis. The proportion of the endocrine tissue composed of insulin- and glucagon-positive areas in CTGF^{e2COIN/e2COIN};Tie-1-Cre (A), CTGF^{e2COIN/e2COIN};Pdx-1-Cre (B), and CTGF^{e2COIN/e2COIN};Ngn3-Cre^BAC (C) mutants at P1. n = 3 animals of each genotype. (D and E) Islet morphogenesis comparison in conditional mutants. Sections from P1 pancreata were immunolabeled with insulin and glucagon (Endo, green) and DBA (red). The distance between the endocrine tissue and ducts was measured in control (D) and mutant (E) pancreata. Although only Pdx-1-Cre mutants are shown, mutants from other Cre were analyzed and found not to be significantly different from controls. (Magnification: 400×.)

Fig. 3.

CTGF overexpression during development led to increased endocrine mass and proliferation. Immunohistochemical analysis of insulin (green) and glucagon (red) in control (A) and bigenic (B) pups at P1 (Magnification: 100×). (C) Quantification of endocrine area at P1 in control (rtTA) and bigenic (rtTA;TetO-CTGF) pups. (D) The number of insulin- and glucagon-positive cells in control and bigenic pups normalized to total pancreatic area. (E) Quantification of Ngn3-positive cells in control and CTGF overexpressing pancreata at E14.5. (F) The ratio of insulin-positive to glucagon-positive cells in control and CTGF overexpressing neonates. (G) The percent of proliferating β cells in control and CTGF overexpressing pups at P1. Representative images of control and bigenic pancreata are shown in G′ and G′′, respectively (Magnification: 400×). Arrowheads indicate proliferating β cells. (H) α-Cell proliferation in control and CTGF overexpressing pups. (I) Quantification of proliferation of MafA-positive cells in control and bigenic pups. n = 3 of each genotype. *P < 0.05 compared with rtTA.

Fig. 4.

Model for CTGF action in endocrine development. Our current and previous studies show that 1, CTGF influences differentiation of delaminating endocrine progenitors to become β cells at the secondary transition. For simplicity, only β cells are shown here. Differentiating β cells begin to produce CTGF. 2, β Cell-derived CTGF acts in an autocrine manner at late gestation to promote replication of MafA⁻/insulin-positive cells (nonnucleated cells), but not MafA⁺ mature β cells (nucleated). 3, In adults, the majority of β cells are MafA⁺ and we hypothesize that these may be refractory to CTGF. 4, Endothelial-derived CTGF may have two roles: an autocrine role for promoting islet vascular development and a paracrine role in stimulating β-cell proliferation.