Elevated transglutaminase 2 activity is associated with hypoxia-induced experimental pulmonary hypertension in mice

Abstract

Previous studies in human patients and animal models have suggested that transglutaminase 2 (TG2) is upregulated in pulmonary hypertension (PH), a phenomenon that appears to be associated with the effects of serotonin (5-hydroxytryptamine; 5-HT) in this disease. Using chemical tools to interrogate and inhibit TG2 activity in vivo, we have shown that pulmonary TG2 undergoes marked post-translational activation in a mouse model of hypoxia-induced PH. We have also identified irreversible fluorinated TG2 inhibitors that may find use as non-invasive positron emission tomography probes for diagnosis and management of this debilitating, lifelong disorder. Pharmacological inhibition of TG2 attenuated the elevated right ventricular pressure but had no effect on hypertrophy of the right ventricle of the heart. A longitudinal study of pulmonary TG2 activity in PH patients is warranted.

Figure 1. Transglutaminase activity and chemical tools for investigating its role in PAH

Transglutaminase 2 (TG2) catalyzes the formation of an isopeptide bond between a protein or peptide substrate harboring a target Gln residue and an acyl acceptor substrate, which may be a Lys residue on another protein or peptide or even a small molecule amine (panel A). One such biogenic amine substrate is serotonin (5-hydroxytryptamine, 5-HT). 5-biotinamidopentylamine (5BP) is another (unnatural) substrate for TG2 that is used as a probe for TG2 activity in tissues (panel B). ERW1041E (Panel C) (1) is an irreversible, acyl-donor substrate-competitive inhibitor of TG2.

Figure 2. Hypoxia induced TG2 activity in mice

C57BL/6 mice were placed in normobaric hypoxic (FIO2 10.5%) chambers for 5–16 days or left in atmosphere as the normoxia-exposed animal controls. TG2 activity in the lung was measured using the nucleophilic substrate, 5BP, as described in the text. To demonstrate that the observed 5BP incorporation was due to TG2 activity, the TG2 inhibitor, ERW1041E, was contemporaneously administered in animals exposed to 16 days of hypoxia. Alexa Fluor 488 (green) was used to visualize TG2 protein, whereas Alexa Fluor 555 (red) corresponded to TG2 activity. (A) Hypoxia induced TG2 activation in lung tissue compared to normoxic treatments. White scale bars are 100[m in length. (B) Quantitation of specific TG2 activity using ImageJ. At least 30 images per condition were collected of which representative images are shown. Data are represented as average +/− standard error. All images were processed identically.

Figure 3. Duration of TG2 blockade in pulmonary hypertensive mice

C57BL/6 mice were placed in normobaric hypoxic (FIO2 10.5%) chambers for 3 weeks or left in atmosphere as the normoxia-exposed animal controls. SU-5416 was given weekly to hypoxia exposed animals, as previously reported in experimental models of pulmonary hypertension (28). TG2 protein and activity in the lung was quantified, as described in the caption to Figure 2. (A) Hypoxia and SU-5416 induced TG2 activation in lung tissue compared to normoxia controls. (B) Quantitative analysis of specific TG2 activity in lungs using ImageJ. (C) The duration of TG2 blockade in response to a single IP injection of 50 mg/kg ERW1041E was estimated. ERW1041E or vehicle was administered 0.5, 6, 18, and 26 hours prior to sacrifice. At least 30 images per condition were collected of which representative images are shown. White scale bars are 100[m in length. (B and D) Data represented as average +/− standard error. All images were processed identically.

Figure 4. Hemodynamic measurements in pulmonary hypertensive mice treated with TG2 inhibitor

C57BL/6 mice were placed in normobaric hypoxic (FIO2 10.5%) chambers for 3 weeks or left in atmosphere as the normoxia-exposed animal controls. SU-5416 was given weekly as described previously (28). Following three weeks of normoxia or hypoxia plus SU-5416, animals were removed to room air and anesthetized. (A) Right ventricular systolic pressure (RVSP) was measured as noted in Methods. Body weights (BW) were recorded. The animals were then euthanized. The heart was dissected and right ventricular (RV) and left ventricular plus septal (LV + S) weights were determined. (B) RV/BW and (C) RV/LV+S ratios were calculated. Values are expressed as means +/− SEM, n= 7 animals in each group. ERW1041E was given once per day (1X) or twice daily (2X) at 50 mg/kg. + p <0.05 vs normoxic; * p < 0.05 vs hypoxic/SU-5416.

Scheme 1

(a) (5a), NMM (3 eq.), DMF ; (a*) (5b), NEt₃ (1 eq.), DMAP (0.1 eq), DMF/DCM 3:2, overnight RT; (b) aq. LiOH, MeOH/THF; (c) EDCI, HOBt, NMM, (S)-DHI, DMF, (d) LiBH₄, EtOH/THF, −78°C; (e) p-nitrophenyl chloroformate, NMM, DCM; (e*) 1,1′-carbonyldiimidazole, ACN, RT; (f) TFA