Transforming growth factor-beta induces cellular injury in experimental diabetic neuropathy

Abstract

The mechanism/s leading to diabetic neuropathy are complex. Transforming growth factor-beta1 (TGF-beta1) has been associated with diabetic nephropathy and retinopathy but not neuropathy. In this study, changes in TGF-beta isoforms were examined in vivo and in vitro. Two groups of animals, streptozotocin diabetic with neuropathy and non-diabetic controls were examined at 4 weeks (n=10/group) and 12 weeks (n=8/group). In diabetic DRG using quantitative real-time PCR (QRT-PCR), TGF-beta1 and TGF-beta2 mRNA, but not TGF-beta3, was increased at 4 and 12 weeks. In sciatic nerve TGF-beta3 mRNA was primarily increased. Immunohistochemistry (DRG) and immunoblotting (sciatic nerve) showed similar differential protein expression. In sciatic nerve TGF-beta formed homo- and hetero-dimers, of which beta(2)/beta(3), beta(1)/beta(1), and beta(1)/beta(3) were significantly increased, while that of the TGF-beta(2)/beta(2) homodimer was decreased, in diabetic compared to non-diabetic rats. In vitro, pretreatment of embryonic DRG with TGF-beta neutralizing antibody prevents the increase in total TGF-beta protein observed with high glucose using immunoblotting. In high glucose conditions, combination with TGF-beta2>beta1 increases the percent of cleaved caspase-3 compared to high glucose alone and TGF-beta neutralizing antibody inhibits this increase. Furthermore, consistent with the findings in diabetic DRG and nerve, TGF-beta isoforms applied directly in vitro reduce neurite outgrowth, and this effect is partially reversed by TGF-beta neutralizing antibody. These findings implicate upregulation of TGF-beta in experimental diabetic peripheral neuropathy and indicate a novel mechanism of cellular injury related to elevated glucose levels. In combination, these findings indicate a potential new target for treatment of diabetic peripheral neuropathy.

Fig. 1. Behavioral assessment of tactile allodynia in 4 week non-diabetic control and STZ-induced diabetic rats using electronic Von-Frey testing

Paw mechanical withdrawal threshold (g) was significantly decreased in diabetic rats. Data is expressed as mean ± SEM. **P<0.001 as compared with non-diabetic control.

Fig. 2. Paw skin biopsy for nerve fibers

Images show sections immunostained with anti-PGP 9.5 antibody in 50 µm cryosections from the hind limb. The arrow indicates intraepidermal nerve fibers; arrow heads indicate dermal nerve fibers (scale bar 30 µm). There is a marked reduction in IENFD in the diabetic rats (B) as compared with the non-diabetic control (A). Lower panel (C) indicates the measurement of IENFD both in non-diabetic and diabetic animals. **P<0.001 as compared to non-diabetic control animals.

Fig. 3. Expression of different isoforms of TGF-β in 4 & 12 week non-diabetic control and STZ-induced diabetic rats in DRG (A – 4 weeks, B – 12 weeks) and sciatic nerve (C – 4 weeks, D – 12 weeks)

QRT-PCR was performed using specific primers as described in experimental procedures. TGF-β1 and TGF-β2, but not TGF-β3 was increased in DRG. In sciatic nerve, only TGF-β3 is increased. Data is expressed as mean ± SEM. *P<0.05 & **P<0.001 as compared with non-diabetic control.

Fig. 4. Immunohistochemistry of 4 weeks non-diabetic control and diabetic rat DRG tissue

(A) Light microscopic images: a, c, e; immunofluoresence (FITC staining) images: b, d, f. TGF-β2 was significantly increased in diabetic DRG (d) compared to non-diabetic control (b) There was no immunofluorescence using pretreatment with pan- TGF-β neutralizing antibody on the section as an internal control (f). (B) Intensity measurement: TGF-β2 staining was significantly increased in diabetic DRG (**P<0.001).

Fig. 5. Western blot analysis of TGF-β isoforms in 4 week non-diabetic and diabetic rat sciatic nerve

(A) Lane 1 & 2 represents non-diabetic protein bands; lane 3 & 4 represents protein bands from diabetic rats. In this blot actin-β was used as an internal control. (B) The density of the relative protein levels were measured and expressed as a graph. Clear bars indicate the relative protein level in non-diabetic controls, black bars indicate the relative protein level in diabetic rats. Data is expressed as mean ± SEM. *P<0.05 as compared with non-diabetic rats.

Fig. 6. Pretreatment with TGF-β neutralizing antibody normalizes the increase in total TGF-β antibody levels with high glucose

Western immunoblot analysis of TGF-β was performed in pooled cultured embryonic DRG neurons with high glucose ± neutralizing antibody: (A) Control DRG neurons (lane 1), high glucose treated neurons (lane 2), high glucose plus TGF-β neutralizing antibody (Lane 3), TGF-β control protein (lane 4). In this blot actin-β was used as internal control. (B) The density of the relative protein levels were measured and expressed as a graph. Data is expressed as mean ± SEM. *P<0.05 as compared with control, ^#P<0.05 as compared with high glucose,

Fig. 7. Increased glucose levels and purified combined TGF-β induces DRG neuronal injury

Purified DRG neurons were treated for 24 hours with high glucose, and/or purified TGF-β protein and stained and counted for caspase-3 cleavage. There is an increase in the percent of caspase-3 positive neurons in DRG neurons exposed to high glucose or TGF-β in a dose dependent manner and there is an additive effect with glucose and higher doses of TGF-β. Data is expressed as mean ± SEM. ***, ^###P<0.01 as compared with control, ⁺⁺⁺P<0.01 as compared with high glucose treatment.

Fig. 8. TGF-β1, -β2, & -β3 increases glucose-induced neuronal injury

(A) A higher percentage of cultured DRG neurons expressed cleaved caspase-3 after incubation in high glucose + TGF-β1, -β2 & -β3 for 24 hours than treated or non-treated controls. Addition of glucose to TGF-β1, -β2 & -β3 had an additive effect on the percent of caspase-3 positive neurons. TGF-β Neutralizing antibody prevented glucose-induced caspase-3 cleavage. Data expressed as mean ± SEM. ^#P<0.05 as compared with non-treated control cells, *P<0.05, **P<0.01 in glucose-treated neurons as compared with the corresponding control condition. (B) DRG neurons that were exposed to high glucose for 48 hours had a higher rate of neuronal injury than non-treated control cells as indicated by TUNEL staining of nuclei for DNA degradation. TGF-β Neutralizing antibody prevented glucose-induced neuronal injury. Data is expressed as mean ± SEM. **P<0.001 in glucose-treated neurons as compared to control.

Fig. 9. TGF-β1, -β2, & -β3 reduce neurite outgrowth

(A) DRG explants cultured with 10 ng/ml of purified TGF-β1, -β2, or -β3 for 48 hours had a reduced mean neurite length compared to control explants. Data expressed as mean ± SEM. (B) high glucose reduces neurite outgrowth but this effect is partially reversed by the use of the TGF-β neutralizing antibody. TGF-β neutralizing antibody alone does not affect neurite growth.