Role For Downstream Effectors Of Ros Injury And Other Newly Discovered Mechanisms

PARP Activation

One of the important effectors of oxidative-nitrosative injury and associated DNA single-strand breakage is activation of the nuclear enzyme PARP (113). Once activated, PARP cleaves nicotinamide adenine dinucleotide (NAD+) with formation of nicoti-namide and ADP-ribose residues, which are attached to nuclear proteins and to PARP itself, with formation of poly(ADP-ribosyl)ated protein polymers. The process leads to:

1. NAD+ depletion and energy failure (26,27,113,114);

2. Changes of transcriptional regulation and gene expression (49,51); and

3. Poly(ADP-ribosyl)ation and inhibition of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase, resulting in diversion of the glycolytic flux toward several pathways implicated in diabetes complications (115).

Recent studies of the author's group (8,26,27) revealed that PARP activation is an early and fundamental mechanism of PDN. It is clearly manifest in peripheral nerve, vasa nervorum, and DRG neurons of STZ-diabetic rats (8,26,27,93) as well as peripheral nerves of STZ-diabetic (94) and ob/ob mice (58). Using endothelial and Schwann cell markers and double immunostaining (8), the author's group localized PARP activation in endothelial and Schwann cells of diabetic rat nerve. The group was first to develop the Western blot analysis of poly(ADP)-ribosylated proteins in rat sciatic nerve (27); using this approach, it was found that poly(ADP)-ribosylated protein abundance increased by 74% in rats with 4-weeks duration of STZ-diabetes in comparison with nondiabetic controls. Furthermore, accumulation of poly(ADP)-ribosylated proteins was found to develop very early, i.e., within about 12-24 hours of exposure of cultured human endothelial and Schwann cells to high glucose (27). PARP-1 protein abundance was not affected by high glucose or PARP inhibitor treatment in either cell type consistent with the current knowledge on PARP-1 as abundantly expressed enzyme with very minor, if any, transcriptional regulation (113).

Studies with several structurally unrelated PARP inhibitors and PARP-deficient (PARP-/-) mice revealed the important role for PARP activation in diabetes-associated MNCV and SNCV deficits, neurovascular dysfunction and peripheral nerve energy failure (8,26). PARP-/- mice were protected from both diabetic and galactose-induced MNCV and SNCV deficits and nerve energy failure that were clearly manifest in the wild-type (PARP+/+) diabetic or galactose-fed mice (8). Two structurally unrelated PARP inhibitors, 3-aminobenzamide and 1,5-ISO, reversed established NBF and conduction deficits as well as energy deficiency in STZ-diabetic rats (8). The third inhibitor, PJ34, essentially corrected nerve conduction deficits and energy deficiency despite relatively modest (17%) reversal of NBF deficit (26). From these observations, it was concluded that PARP inhibition counteracts diabetes-induced changes in nerve energy state (the variable that correlates best with nerve conduction) primarily through recently discovered effect on the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (115) and resulting improvement of glucose utilization in Schwann cells. The latter is consistent with normalization of free NAD+/NADH ratio, an index of tricarboxylic cycle activity and glucose utilization, in the peripheral nerve mitochondrial matrix of PJ34-treated diabetic rats (26). In the same study, PJ34 treatment counteracted accumulation of lactate and glutamate, as well as depletion of a-glutarate in diabetic peripheral nerve. Nerve glucose and sorbitol pathway intermediate concentrations were similarly elevated in PJ34-treated and untreated rats (26), which is consistent with the downstream localization of PARP activation, consequent to increased AR activity and resulting oxidative-nitrosative stress, in the pathogenesis of diabetic complications. In other two studies of the author's group, diabetes-induced peripheral nerve protein poly(ADP)-ribosylation was blunted by the ARI fidarestat and the peroxynitrite decomposition catalyst FP15 (45,94), thus, indicating the importance of increased AR activity and nitrosative stress among the upstream mechanisms underlying PARP activation.

The low-dose PARP inhibitor-containing combination therapies (with two vasodilators, the ACE-inhibitor lisinopril and P-adrenoceptor agonist salbutamol), reversed neurovascular dysfunction, SNCV deficit (MNCV deficit was reversed by salbutamol-, but not lisinopril-containing drug combination) as well as thermal and mechanical hyperalgesia in rats with short-term STZ-diabetes (27). Theoretically, PARP activation can contribute to diabetic neuropathic pain and abnormal sensory responses through several mechanisms including, but not limited to:

1. Upregulation of TNF-a and other inflammatory genes;

2. Activation of p38 MAP kinase in the spinal cord and Schwann cells; and

3. Ca2+-regulated excitotoxic insults, all of which have been implicated in the pathogenesis of painful neuropathy (116-118).

PARP-dependence of TNF-a overexpression, p38 MAP kinase activation, and Ca2+-regulated excitotoxic insults in pathological conditions associated with oxidative stress have been experimentally documented (113,119-121). Furthermore, recent studies from the author's group revealed that PARP activation not only results from, but also exacerbates oxidative-nitrosative stress in peripheral nerve, vasa nervorum, and human Schwann cells (122). Detailed studies of the role for PARP activation in diabetic neuropathic pain are in progress in the laboratory.

Activation of MAPKs

Numerous findings indicate that ROS and reactive nitrogen species cause MAPK activation (17,123), and increasing evidence supports the importance of MAPKs in the pathogenesis of PDN. ERK, p38 MAPK, and JNK are activated in DRG neurons of STZ-diabetic rats (17). Enhanced p38 MAPK activation in response to endothelin-1 or platelet-derived growth factor has been observed in immortalized Schwann cells cultured in 25 mM glucose in comparison with those in 5 mM glucose (124). Sural nerve JNK activation and increases in total levels of p38 and JNK have been observed in patients with both type 1 and type 2 diabetes (17). MAPKs are implicated in aberrant neurofilament phosphorylation, a phenomenon involved in the etiology of the diabetic sensory polyneu-ropathy (125). Fernyhough et al. (125) have reported a two- to threefold elevation of neurofilament phosphorylation in lumbar DRG of STZ-diabetic and spontaneously diabetic BB rats as well as 2.5-fold elevation in neurofilament M phosphorylation in sural nerve of BB rats. Diabetes-induced three- to fourfold increase in phosphorylation of a 54-kDa isoform of JNK in DRG and sural nerve correlated with elevated c-Jun and neurofilament phosphorylation. p38 activation in DRG neurons of STZ-diabetic rats is prevented by the ARIs sorbinil and fidarestat, which suggests the important role of AR in diabetes-related alterations in MAPK signaling (126). The p38 MAPK inhibitor SB239063 corrected MNCV and SNCV deficits in STZ-diabetic rats, thus implicating p38 MAPK in motor and sensory nerve dysfunction (47,126). In the same animal model, the p38-a MAPK inhibitor SD-282 counteracted mechanical allodynia, C-, but not a delta-fiber-mediated thermal hyperalgesia, and attenuated flinching behavior during the quiescent period and the second phase of the formalin response (127). Spinal p38 MAPK has also been implicated in the neuropathic pain induced by inflammation (128).

Activation of NF-kB

Both PARP-1 and MAPKs are involved in transcriptional regulation of gene expression, through the transcription factors NF-kB, activator protein-1, p53, and others (113,114,129). Activated NF-kB has been identified in perineurium, epineurial vessels, and endoneurium in sural nerve biopsies of subjects with impaired glucose tolerance (76) and overt diabetes (68). NF-kB activation was also found in isolated Schwann cells cultured in high glucose medium in comparison with those in low glucose (130) and such activation was prevented by the ARI fidarestat. Similar effect of AR inhibition on NF-kB activation was observed in high glucose-exposed vascular smooth muscle cells (52). Activation of NF-kB and other transcription factors by high glucose (52,130) and oxidative stress (131) leads to upregulation of inducible nitric oxide synthase, COX-2, endothelin-1, cell adhesion molecules, and inflammatory genes (49,51). Growing evidence indicates that the aforementioned transcription factors and their target genes are involved in the pathogenesis of diabetic complications, and, in particular, PDN (9,10, 68,132). Thus, it is not surprising that inhibition of NF-kB by pyrrolidine dithiocarba-mate and the serine protease inhibitor V-a-tosyl-L-lysine chloro-methylketone, i.e., IkB protease activity-blocking agent, corrected nerve conduction and blood flow deficits in STZ-diabetic rats (9). In the same animal model, V-a-tosyl-L-lysine chloro-methylketone also partially reversed gastric autonomic neuropathy (9).

Activation of COX-2 and 12/15-LO

Evidence for the important role of arachidonic acid metabolic pathways, i.e., COX-1 and COX-2, cytochrome P450 epoxygenase, and LOs in diabetes complications is emerging. The lipid products of these pathways include thromboxane, prostaglandins, leukotrienes, lipoxins, epoxyetraenoic acid, 12-hydroperoxy-eicosatetraenoic acid, 12-(HETE) hydroxy-eicosatetraenoic acid, 15-HETE, and a whole variety of their derivatives. COX-1 protein expression was reported unchanged in the diabetic peripheral nerve (10) and reduced in the spinal cord (133), whereas COX-2 protein abundance was increased in both tissues (10,133). Selective COX-2 inhibition with meloxicam prevented motor nerve conduction and endoneurial nutritive blood flow deficits in the diabetic rats (10). The same group (134) found diabetic COX-2 deficient mice protected from MNCV and SNCV slowing that was clearly manifest in the diabetic wild-type mice. Several studies implicate COX-2 overexpression in increased production of hydroxyl radicals and hydrogen peroxide as well as lipid peroxidation (110,135), and STZ-diabetic COX-2-/- mice have been reported to develop less severe peripheral nerve oxidative stress than the diabetic wild-type mice (134).

One of the most interesting members of the LO family is 12/15-LO, a nonheme iron-containing dioxygenase that forms 12-hydroperoxy-eicosatetraenoic acid and 12- and 15-HETEs and oxidizes esterified arachidonic acid in lipoproteins (cholesteryl esters) and phospholipids (109,136). 12/15-LO is abundantly expressed in endothelial cells (i.e., aortic and retinal endothelial cells), smooth muscle cells, monocyte/macrophages as well as renal mesangial cells, tubular epithelial cells, and podocytes, and the enzyme expression is increased under diabetic and hyperglycemic conditions (109,136).

Recent in vivo and cell culture studies (109,136,137) revealed that high glucose-induced 12/15-LO activation affects multiple metabolic and signal transduction pathways, transcriptional regulation, and gene expression. The major consequences include increased free radical production and lipid peroxidation, MAPK and NF-kB activation, inflammatory response, excessive growth as well as adhesive and chemoattractant effects (109,136,137). Recent findings from the author's group suggest an important role for 12/15-LO activation in peripheral DN (14). In particular, it was found that:

1. 12/15-LO is abundantly expressed in mouse peripheral nerve and its expression increases in diabetic conditions;

2. 12/15-LO-/- mice with both type 1 (streptozotocin-induced) and type 2 (high fat diet-induced) diabetes develop less severe peripheral DN than wild-type mice;

3. Some manifestations of peripheral DN in STZ-diabetic mice are reversed by a short-term 12/15-LO inhibitor treatment;

4. 12/15-LO is abundantly expressed in human Schwann cells (HSC), one of the major cell targets in human DN, and its overexpression is clearly manifest after a short-term (24 h) exposure to high glucose; and

5. 12/15-LO overexpression in high glucose-exposed HSC contributes to activation (phosphorylation) of all three subtypes of MAPKs including p38 MAPK, ERK V2, and JNK-1, recently implicated in the pathogenesis of both experimental and human DN (17,47,125-127).

These results provide the rationale for detailed studies of the role for 12/15-LO pathway in diabetic neuropathic changes.

Altered Ca2+ Homeostasis and Signaling

Growing evidence suggests that diabetic sensory neuropathy is associated with abnormal Ca2+ homeostasis and signaling in DRG neurons (138,139). Enhanced Ca2+ influx through multiple high-threshold calcium currents is present in sensory neurons of several rat models of diabetes mellitus, including the spontaneously diabetic BioBred/ Worchester (BB/W) rats and STZ-diabetic rats (139). Sensory neurons of STZ-diabetic rats have increased mRNA expression of voltage-gated calcium channels (140). Enhanced calcium entry in diabetes is also linked to the impairment of G protein-coupled modulation of calcium channel function (138). Increased AR activity and taurine deficiency contribute to diabetes-related enhancement of voltage-dependent calcium currents in DRG neurons (138,139). These two mechanisms probably converge at the level of oxidative stress and result in PARP activation known to promote [Ca2+]i accumulation and impair calcium signaling. Resting [Ca2+] increased in rat lumbar nociceptive neurons with the duration of STZ diabetes, whereas calcium mobilization from the endoplasmic reticulum reduced (140). [Ca2+] is essentially required for 12/15-LO catalytic activity (141) predominantly confined to cytoplasm (142).

Evidence for important role of [Ca2+]i in neuropathic pain of different origin and in particular, diabetic neuropathic pain is emerging. Luo et al. (143) found that injury to type-specific calcium channel alpha 2delta-1 subunit upregulation in rat neuropathic pain models (mechanical nerve injuries, diabetic neuropathy, chemical neuropathy) correlated with antiallodynic effect of the anticonvulsant gabapentin. Furthermore, a recent 6-week, randomized, double-blind, multicenter clinical trial in 246 patients with painful diabetic neuropathy revealed that pregabalin, a new drug that interacts with the alpha 2delta-1 protein subunit of the voltage-gated calcium channel, is a efficacious and safe treatment for diabetic neuropathic pain (144). Similar findings have been obtained in another 12-week, randomized, double-blind, multicenter, placebo-controlled trial of pregabalin that revealed a significant pain relief in patients with chronic postherpetic neuralgia or painful diabetic neuropathy (145).

Activation of NHE-1

Recent studies from the author's group suggest an important role for NHE-1 in PDN (146). The NHE-1 specific inhibitor cariporide, at least, partially prevented MNCV and SNCV deficits, thermal hypoalgesia, mechanical hyperalgesia, and tactile allodynia in

STZ-diabetic rats. STZ-diabetic NHE-1+/- mice developed less severe early PDN than the wild-type mice. Increased NHE-1 protein expression was found early (about 24 hours) after exposure of human endothelial and Schwann cells to high glucose, thus suggesting that this mechanism can be of importance in human PDN (147).

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