than to hyperglycemia and can be blocked by the administration of either d-a-tocopherol (a nonspecific PKC inhibitor) or ruboxistaurin (RBX) (a PKCβ-specific inhibitor).

BASEMENT MEMBRANE AND ECM CHANGES

Diabetic vasculature undergoes other early changes in the course of diabetes. Increased deposition of ECM causes capillary basement membrane thickening (42). This in turn affects vascular permeability, cellular adhesion, proliferation, differentiation, and gene expression (43). Increased expression of collagens, fibronectin, and laminin has been reported prior to basement membrane thickening (44, 45). Expression and transcription of these substances have been shown to be reduced in response to administering PKC inhibitors.

Growth factors are also thought to play a role in basement membrane and ECM alterations. For example, transforming growth factor β (TGFβ) has been established as a regulator of ECM accumulation and increases expression of certain collagens and fibronectin. Studies show increased expression of TGFβ in response to hyperglycemia (46). Another growth factor, connective tissue growth factor (CTGF), has also been shown to regulate ECM accumulation via TGFβ-dependent and TGFβ-independent mechanisms (47). Hyperglycemia-induced increases in the expression of CTGF appear to be dependent on both TGFβ expression and the PKC signaling pathway, as increases are tempered by an anti-TGFβ antibody and by PKC inhibition (48–49). PKC activation may play a role in this increase leading to the accumulation of ECM. Certain protooncogenes, c-fos and c-jun, are induced by PKC. They regulate gene expression via an AP-1 binding site whose consensus sequence is present in the promoter region of TGFβ, CTGF, fibronectin, and laminin (50–53).

VASCULAR PERMEABILITY AND ANGIOGENESIS

Diabetes causes a marked increase in vascular permeability to macromolecules such as albumin (54). This has been noted significantly in the retinal and renal vasculature. This change in permeability in the retinal vasculature leads to the clinical sequelae of transudation of fluid into the retina and subsequent visual loss from macular edema. Cultured endothelial cells have shown increased permeability in response to phorbol ester-activated PKC to macromolecules including albumin (55–56). This increase is reduced by PKC inhibitors. It has been suggested that PKC causes the phosphorylation of certain cytoskeletal proteins (caldesmon, vimentin, talin, and vinculin), and through this mechanism, stimulates the endothelial cell contractile apparatus, resulting in increased vascular permeability (57–59).

Increased levels of vascular endothelial growth factor (VEGF) have been demonstrated in vitreous fluid and aqueous in patients with proliferative diabetic retinopathy (60). VEGF has mitogenic effects on endothelial cells and promotes vascular permeability. It is also a key factor in mediating hypoxia-induced angiogenesis. Increased expression of VEGF is reported in vascular smooth muscle cells in response to high glucose. PKC inhibition leads to a decrease in this increased VEGF expression. The nonisoform-specific PKC inhibitors GFX and H-7 prevent cellular proliferation in response to VEGF. In addition, LY333531 (ruboxistaurin), which selectively inhibits the PKCβ isoform, also decreases VEGF’s mitogenic effects (versus the antisense PKCα oligonucleotide that did

not reduce the mitogenic effects). This suggests an important role for PKCβ in VEGFmediated cellular proliferation (61, 62).

INHIBITION OF PKCβ

Many early PKC inhibitors were nonspecific and were associated with a variety of adverse clinical side effects. More recent PKC inhibitors proposed for clinical study, including ruboxistaurin (RBX), which inhibits PKCβ, have targeted specific PKC isoforms. The PKCβ isoform is of particular clinical interest because it demonstrates increased activation in many vascular tissues, including the eye and kidney, in the diabetic state. RBX exerts its inhibition on the cPKC family, and more specifically has affinity for PKCβI and PKCβII over PKCα and other PKC isoforms (63). In addition, in a specific dose range, it demonstrates selective inhibition of PKC over other kinases such as calcium-calmodulin and src-tyrosine kinases (63).

When given orally to diabetic rats, RBX increases retinal blood flow, and improves glomerular filtration rates and albumin excretion (64). It has also been shown to attenuate the microvascular flow disturbances caused by leukocyte adhesion (65). Further studies using intravitreal administration of RBX in diabetic rats showed decreased PKC activation and increased retinal blood flow (37). RBX also suppresses VEGF-mediated retinal vascular permeability in vivo (62) and prevents retinal neovascularization development in a pig model of ischemic retinal disease (66). A recent study demonstrated that RBX was well tolerated by diabetic patients in doses up to 16 mg twice daily for 28 days. At these doses, it decreased diabetes-induced retinal circulation time abnormalities without any significant safety issues (67). Subsequent phase 3 studies demonstrated that 32 mg of oral RBX given once daily over 3 years significantly reduced the rate of sustained moderate visual loss (68). In addition, initial macular laser treatment was 26% less frequent in patients on RBX compared to placebo (p = 0.0008), and macular edema progressed significantly less frequently to within 100 µm of the fovea (68). To date, 11 clinical trials of this drug have been completed or are currently recruiting that evaluate RBX’s additional effects on endothelial dysfunction, peripheral neuropathy, and nephropathy in diabetic patients (69). Clinical trials involving RBX are discussed in further detail in Chap. 18.

CONCLUSIONS

Diabetic complications involving the eye, kidney, heart, and nerve all involve activation of the DAG-PKC pathway. Hyperglycemia increases the activity of this pathway, either directly or indirectly via oxidants and glycated products. PKC inhibition has been shown to ameliorate many of hyperglycemia’s adverse effects on the vasculature, including changes in retinal blood flow, thickening of basement membrane and extracellular matrix, and increases in vascular permeability and angiogenesis. Given the presence of multiple PKC isoforms each with specific triggers and actions, the need for targeted therapy is crucial to prevent complications. RBX preferentially inhibits the PKCβ isoform and is well tolerated by diabetic patients. However, it is very likely that the activation of other PKC isoforms also causes significant retinal pathologies and will have to be inhibited in order to stop the progression of diabetic retinopathy.

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