MT-100, a human Tie2-agonistic antibody, improves penile neurovasculature in diabetic mice via the novel target Srpx2

Abstract

Diabetes is an incurable, chronic disease that can lead to many complications, including angiopathy, peripheral neuropathy, and erectile dysfunction (ED). The angiopoietin-Tie2 signaling pathway plays a critical role in blood vessel development, formation, remodeling, and peripheral nerve regeneration. Therefore, strategies for activating the Tie2 signaling pathway have been developed as potential therapies for neurovascular diseases. Here, we developed a human Tie2-agonistic antibody (MT-100) that not only resists Ang-2 antagonism and activates Tie2 signaling but also regulates a novel target, sushi repeat-containing protein X-linked 2 (Srpx2). This regulation led to the survival of vascular and neuronal cells, a reduction in the production of reactive oxygen species (ROS), activation of the PI3K/AKT/eNOS signaling pathway, increased expression of neurotrophic factors, and ultimately alleviation of ED in diabetic mice. Our findings not only provide conclusive evidence that MT-100 is a promising therapeutic strategy for the treatment of diabetic ED but also suggest it has substantial clinical applications for other complications associated with diabetes.

Fig. 1. Generation and characterization of a cross-species Tie2-agonistic antibody.

a Screening of Tie2-binding scFv antibodies. Three rounds of phage display panning were performed with hTie2-ECD-Fc, and individual scFvs were screened from the third-round output. The cross-species binding activity of the screened scFvs was evaluated using hTie2-ECD-Fc or mTie2-ECD-Fc via ELISA. b Phospho-RTK arrays in HUVECs. MT-100 was produced as an IgG and incubated with HUVECs. Cell lysates obtained before or after MT-100 treatment were used for RTK arrays. c Affinity measurement via BLI. Recombinant hTie2-ECD was immobilized on a biosensor tip, and the indicated concentrations of MT-100 were allowed to bind to the tip. The K_D (K_off/K_on) value was calculated via global fitting analysis. d Assessment of cross-species binding of MT-100. The full-length Tie2 of each species was transiently expressed in HEK-293T cells, and the binding of MT-100 was analyzed by flow cytometry. e Evaluation of Tie2 and cascade signaling activation in HUVECs. The indicated concentrations of MT-100 were incubated with HUVECs, and phosphorylated Tie2 was detected in the lysates of the cells via immunoprecipitation with an anti-Tie2 antibody. Phosphorylated eNOS, AKT, and ERK were detected in whole-cell lysates (WCLs). f Evaluation of Tie2 and cascade signaling activation in a mouse cell line. 3T3-J2 cells were treated with various concentrations of MT-100, and phosphorylated Tie2, AKT, and ERK were detected in WCLs. g Evaluation of the ability of MT-100 to block the Ang-2 and Tie2 interaction. Recombinant hTie2-ECD was immobilized on a biosensor, and buffer (red) or MT-100 (blue) was incubated with the biosensor. Ang-2 was further allowed to bind to each biosensor, and the binding kinetics were analyzed via biolayer interferometry. Illustrations were created with BioRender.com. h Assessment of endothelial cell integrity. HUVECs cultured under confluent conditions were incubated with the indicated treatments. Tight junction integrity was validated by VE-cadherin staining. Nuclei were stained with DAPI (blue). Scale bars, 100 µm. i Effect of MT-100 on endothelial cell survival. The indicated concentrations of MT-100 were incubated with HUVECs under hypoxic and serum-free conditions. Ang-1 was used as a positive control. Cell survival was analyzed via trypan blue staining. The data are presented as mean ± SEM (n = 3). The relative ratio in the control was set to 100. **P < 0.01. DAPI, 4,6-diamidino-2-phenylindole; HUVECs, human umbilical vein endothelial cells.

Fig. 2. MT-100 induces neurovascular regeneration and improves erectile function under diabetic conditions.

a Tube formation assay. HUVECs were treated with phosphate-buffered saline (PBS), negative control IgG1 (NC, 10 μg/mL), or MT-100 (1 μg, 10 μg, or 20 μg/mL) under normal glucose (NG) or high glucose (HG) conditions for 3 days. Scale bars, 100 µm. b Quantification of the number of master junctions per field (n = 4). c Migration of HUVECs 24 h after treatment with PBS, NC (10 μg/mL), or MT-100 (10 μg/mL) under NG or HG conditions for 3 days. Scale bars, 100 µm. d The ratio of migrated HUVECs in the frame line was quantified (n = 4). e Representative intracavernous pressure (ICP) responses of age-matched control and STZ-induced diabetic (DM) mice stimulated at 2 weeks after repeated intracavernous injection (days −3 and 0) of PBS (20 μL), NC (10 μg in 20 μL of PBS), or MT-100 (1 μg, 10 μg, or 20 μg in 20 μL of PBS) on days 0 and 3. The cavernous nerve was stimulated at 5 V. The stimulus interval is indicated by a solid bar. f, g Ratios of the mean maximal ICP (f) and total ICP (g, area under the curve) to mean systolic blood pressure were calculated for each group (n = 7). h, i Representative images of immunofluorescence staining of corpus cavernosum (h) and dorsal nerve bundle (i) tissues from age-matched control and DM mice 2 weeks after repeated intracavernous injection (days −3 and 0) of PBS (20 μL), NC (10 μg in 20 μL of PBS), or MT-100 (10 μg in 20 μL of PBS) for NG2 (red), CD31 (green), NF (red), or nNOS (green) after ICP studies. Nuclei were stained with DAPI (blue). Scale bars, 100 µm (h) and 25 µm (i). j‒m Quantitative analysis of corpus cavernosum pericyte (NG2), endothelial cell (CD31), and dorsal nerve bundle neuronal cell (NF and nNOS) content using ImageJ software. The data are presented as mean ± SEM (n = 4). The relative ratio in the control or NG groups was arbitrarily set to 1. *P < 0.05; **P < 0.01; ***P < 0.001. DAPI 4,6-diamidino-2-phenylindole, HUVECs human umbilical vein endothelial cells, N.S. not significant.

Fig. 3. MT-100 increases proliferation and decreases apoptosis of corpus cavernosum endothelial cells under diabetic conditions.

a Representative images of immunofluorescently stained corpus cavernosum tissues from age-matched control and STZ-induced diabetic (DM) mice 2 weeks after repeated intracavernous injection (days −3 and 0) of phosphate-buffered saline (PBS, 20 μL), negative control IgG1 (NC, 10 μg in 20 μL of PBS), and MT-100 (10 μg in 20 μL of PBS) for phospho-histone H3 (PH3; red) and CD31 (green) after ICP studies. b TUNEL assay (green) and CD31 (red) immunofluorescence staining under the same conditions described above. Nuclei were stained with DAPI (blue). Scale bars, 100 µm. c, d Number of PH3-positive (c) and TUNEL-positive (d) endothelial cells quantified by ImageJ software. The results are presented as mean ± SEM (n = 4). e–h In vitro studies of the proliferation assay (PH3) (e) and TUNEL assay (f) in HUVECs treated with PBS, NC (10 μg/mL), or MT-100 (10 μg/mL) under normal glucose (NG) or high glucose (HG) conditions for 3 days. Nuclei were stained with DAPI (blue). Scale bars, 25 µm. g, h Number of PH3-positive (g) and TUNEL-positive (h) HUVECs quantified via ImageJ software. The results are presented as mean ± SEM (n = 4). **P < 0.01; ***P < 0.001. DAPI 4,6-diamidino-2-phenylindole, TUNEL terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling, HUVECs human umbilical vein endothelial cells, N.S. not significant.

Fig. 4. MT-100 reduces permeability under diabetic conditions.

a Claudin-5 (green) and occludin (red) immunofluorescence staining of corpus cavernosum tissues from age-matched control and STZ-induced diabetic (DM) mice 2 weeks after repeated intracavernous injection (days −3 and 0) of phosphate-buffered saline (PBS, 20 μL), negative control IgG1 (NC, 10 μg in 20 μL of PBS), or MT-100 (10 μg in 20 μL of PBS). Nuclei were stained with DAPI (blue). Scale bars, 100 µm. b, c Quantitative analysis of claudin-5- and occludin-immunopositive areas using ImageJ software. The data are presented as mean ± SEM (n = 4). The relative ratio in the control groups was arbitrarily set to 1. *P < 0.05; **P < 0.01; ***P < 0.001. d The permeability assay was performed by measuring the leakiness of Evans blue (EB)-albumin in HUVECs treated with NC (10 μg in 20 μL of PBS) or MT-100 (10 μg in 20 μL of PBS) under normal glucose (NG) or high glucose (HG) conditions for 3 days. Clearance of EB-albumin was measured every 30 min up to 90 min. Data are presented as mean ± SEM (n = 4). **P < 0.01; ***P < 0.001. DAPI 4,6-diamidino-2-phenylindole, HUVECs human umbilical vein endothelial cells, N.S. not significant.

Fig. 5. MT-100 decreases cavernous ROS production in diabetic mice.

a In situ detection of superoxide anion (hydroethidine, red) and nitrotyrosine production (green) in corpus cavernosum tissues from age-matched control and STZ-induced diabetic (DM) mice 2 weeks after repeated intracavernous injection (days −3 and 0) of phosphate-buffered saline (PBS, 20 μL), negative control IgG1 (NC, 10 μg in 20 μL of PBS), or MT-100 (10 μg in 20 μL of PBS). Nuclei were stained with DAPI (blue). Scale bars, 100 µm. b, c Quantitative analysis of the ethidium bromide fluorescence-immunopositive cavernosum area (b) and nitrotyrosine-immunopositive cavernosum area (c) using ImageJ software. The data are presented as mean ± SEM (n = 4). The relative ratio in the control groups was arbitrarily set to 1. *P < 0.05; **P < 0.01; ***P < 0.001. DAPI 4,6-diamidino-2-phenylindole, ROS reactive oxygen species, N.S. not significant.

Fig. 6. MT-100 improves erectile function via a novel target, Srpx2, in diabetic mice.

a Representative Western blot for Adam8, Hmox1, and Srpx2 in corpus cavernosum tissues from age-matched control and STZ-induced diabetic (DM) mice 2 weeks after repeated intracavernous injection (days −3 and 0) of phosphate-buffered saline (PBS, 20 μL), negative control IgG1 (NC, 10 μg in 20 μL of PBS), or MT-100 (10 μg in 20 μL of PBS). b‒d Band intensity values for each neurotropic factor normalized to the density of β-actin and quantified using ImageJ software. The data are presented as mean ± SEM (n = 4). The relative ratio in the control group was defined as 1. e Representative intracavernous pressure (ICP) responses of age-matched control and DM mice stimulated after 2 weeks under the indicated conditions. The cavernous nerve was stimulated at 5 V. The stimulus interval is indicated by a solid bar. f, g Ratios of the mean maximal ICP (f) and total ICP (g, area under the curve) to the mean systolic blood pressure were calculated for each group (n = 7). h, i Representative images of immunofluorescence staining of corpus cavernosum (h) and dorsal nerve bundle (i) tissues for NG2 (red), CD31 (green), NF (red), and nNOS (green) after ICP studies. Nuclei were stained with DAPI (blue). Scale bars, 100 µm (h) and 25 µm (i). j‒m Quantitative analysis of corpus cavernosum pericyte (NG2), endothelial cell (CD31), and dorsal nerve bundle neuronal cell (NF and nNOS) content using ImageJ software. The data are presented as mean ± SEM (n = 4). The relative ratio in the control or normal glucose groups was arbitrarily set to 1. *P < 0.05; **P < 0.01; ***P < 0.001. DAPI 4,6-diamidino-2-phenylindole, N.S. not significant.

Fig. 7. Identification of the mechanism by which MT-100/Srpx2 restores erections in diabetic mice.

a, b Representative images of immunofluorescence staining of tight junction proteins (a, claudin-5 and occludin) and ROS production (b, hydroethidine and nitrotyrosine) under the indicated conditions. c Representative Western blot for p-PI3K^Tyr199, p-AKT^Ser473, NT3, BDNF, and NGF in corpus cavernosum tissues from the indicated groups. d–l Quantitative analysis of tight junction proteins (d, e), ROS production (f, g), and Western blots (h–l) using ImageJ software. The data are presented as mean ± SEM (n = 4). The relative ratio in the control or normal glucose groups was arbitrarily set to 1. *P < 0.05; **P < 0.01; ***P < 0.001. DAPI 4,6-diamidino-2-phenylindole, BDNF brain-derived neurotrophic factor, NGF nerve growth factor, NT-3 neurotrophin-3, DM STZ-induced diabetes, N.S. not significant.

Fig. 8

Schematic of the proposed mechanism by which MT-100 improved erectile function in diabetic mice.