**6.1. Therapeutic approaches targeting the mitochondria**

Glycemic control has been the main therapeutic modality for prevention and treatment of hyperglycemic injury and diabetes complications. However, recent studies confirmed that glycemic control provides limited protection against the cardiovascular events, and adjunct therapy is necessary to reduce the risk of complications. Previous efforts found aldose reductase (a key enzyme of the polyol pathway) to serve as potential drug target and found that inhibition of aldose reductase may prevent the pathological changes that occur in response to high sorbitol levels [141]. The results of the clinical trials were less impressive than expected from preclinical studies, still the first aldose reductase inhibitor has been marketed in Asia for the treatment of diabetes complications [142].

was identified as a selective inhibitor of ROS production by the ubiquinone-binding site of complex I, and S3QELs ("sequels," selective suppressors of site IIIOQ electron leak) were found to act as inhibitors of the outer ubiquinone-binding site similar to terpestacin [156– 158]. Unfortunately, there are no data about their action against hyperglycemia-induced ROS production. Statins also block the mitochondrial ROS production but they simultaneously reduce the mitochondrial respiration and may cause toxicity [38, 151]. A further inhibitor of

stimulator of mitochondrial metabolism at low concentrations via electron donation [106].

Finally, many of the currently used anti-diabetic medications also possess mitochondrial targets and they might be partly responsible for their beneficial effects on endothelial cells and the vasculature. The biguanide metformin apart from activating AMPK also acts as a mild and transient inhibitor of respiratory complex I [161]. The sulfonylurea glibenclamide inhibits the mitochondrial ATP-sensitive potassium channel (mitoKATP), decreases the mitochondrial membrane potential and ROS production and increases the respiration rate [162, 163]. Thiazolidinediones (TZDs) also possess a specific mitochondrial target (mTOT, mitochondrial target of TZDs), which comprise a recently identified protein complex. TZDs bind to a pyruvate carrier complex in the mitochondria and modulate the pyruvate entry into the mitochondria that may explain their antioxidant effect [164]. The discovery of these novel mitochondrial targets is expected to promote target-based drug discovery efforts and may

Endothelial cells exposed to oxidative stress and the proinflammatory environment undergo accelerated aging in hyperglycemia, thus stimulation of cell replacement with fresh endothelial cells may help restore the vascular function [126]. While it is difficult to estimate the exact number of endothelial cells, a reduction of progenitor cell count and functionality was detected in diabetes, and it is expected to result in a decreased number of functional endothelial cells in the vasculature [24, 127]. Even in the retina, where neovascularization (proliferative diabetic retinopathy) is the characteristic event, progenitor cell therapy is expected to cause improvement and treat the ischemic vascular abnormalities [165]. Currently, it is difficult to predict which treatment strategy will prove effective in diabetes, but probably the drug-induced enhancement of progenitor cell potency has the highest translational potential in diabetes [166, 167]. Drugs that were suggested for progenitor cell enhancement include statins, losartan (angiotensin II receptor antagonist), aliskiren (direct renin inhibitor), hydrogen sulfide, thymosin β4 and the CXCR4 (C-X-C chemokine receptor type 4) antagonist plerixafor (AMD3100, 1,1-[1,4-Phenylenebis(methylene)]bis-1,4,8,11-tetraazacyclotetradecane) [167]. As some of these drugs are in use in diabetic patients, an evaluation of the effect of these compounds on diabetic progenitor cells might be available in the near

S supplementation either using sodium sulfide or mitochondria-targeted donor molecules inhibits the hyperglycemia-induced ROS production and endothelial dysfunction at low con-

S, a known inhibitor of complex IV, that turns out to act as

S is effective against diabetic nephropathy

Hyperglycemia-Induced Endothelial Dysfunction http://dx.doi.org/10.5772/intechopen.71433 197

mitochondrial respiration is H2

centrations [35, 94]. Long-term administration of H2

provide new compounds in the upcoming years.

**6.2. Therapeutic approaches to replace the injured endothelial cells**

and retinopathy in animal models [159, 160].

H2

Following the discovery that mitochondrial superoxide is responsible for the induction of all ROS-producing pathways in hyperglycemic endothelial cells, an intense search began for mitochondrial drug targets and inhibitors of mitochondrial superoxide generation [26, 83, 84]. Mitochondria-specific targeting moieties have been developed and linked to antioxidants or superoxide dismutase (SOD) mimetics [143, 144]. The majority of these molecules use the triphenylphosphonium (TPP) targeting group and attain 100- to 500-fold accumulation in the mitochondria [145, 146]. Both the mitochondria-targeted ubiquinol (MitoQ) and piperidine nitroxide TEMPO (mitoTEMPO) proved beneficial in diabetes models [147–149]. Overexpression of MnSOD was found to protect against diabetic retinopathy in a transgenic model, but long-term delivery of MnSOD is unresolved in humans [150].

While no protein target has been identified, various phenotypic screens were performed, which allowed the target-agnostic discovery of potential drug candidates [38, 151]. In cellbased models, paroxetine emerged as a mitochondrial free radical scavenger and glucocorticoid steroids as UCP2 inducers in hyperglycemic endothelial cells [35, 38]. In another screen, which also used multiple assay variables as output and examined the OXPHOS-associated gene expression, the antihelmintic drug mebendazole and the Chinese herbal medicine deoxysappanone B emerged as inhibitors of mitochondrial ROS production [151]. The protective effect of all of these drugs might be a direct consequence of the normalization of the mitochondrial membrane potential or free radical scavenging.

The mitochondrial antioxidant effect of select microtubular drugs, which was independently confirmed by the previous two studies, might be related to their mitochondrial fusion promoting effect, since excessive mitochondrial fission and fragmentation occur in hyperglycemia [38, 151]. To identify compounds that promote mitochondrial fusion, a separate phenotypic screening program was conducted [152]. This study identified hydrazone M1, a small molecule that restore the mitochondrial network in cells but its efficacy has not been tested in diabetes [152, 153]. Also, compounds that block the mitochondrial fission are expected to improve the mitochondrial function and reduce the mitochondrial ROS production in hyperglycemia, but these inhibitors (mitochondrial division inhibitor-1 (mdivi-1), dynasore, P110 and 15-oxospiramilactone) have not been tested in diabetes [154, 155].

In another effort to identify compounds that reduce the mitochondrial ROS production but do not interfere with energy production, isolated mitochondria were used for high throughput screening and hit compounds were selected based on a dual output of ROS production and respiration rate [156, 157]. CN-POBS (N-cyclohexyl-4-(4-nitrophenoxy)benzenesulfonamide) was identified as a selective inhibitor of ROS production by the ubiquinone-binding site of complex I, and S3QELs ("sequels," selective suppressors of site IIIOQ electron leak) were found to act as inhibitors of the outer ubiquinone-binding site similar to terpestacin [156– 158]. Unfortunately, there are no data about their action against hyperglycemia-induced ROS production. Statins also block the mitochondrial ROS production but they simultaneously reduce the mitochondrial respiration and may cause toxicity [38, 151]. A further inhibitor of mitochondrial respiration is H2 S, a known inhibitor of complex IV, that turns out to act as stimulator of mitochondrial metabolism at low concentrations via electron donation [106]. H2 S supplementation either using sodium sulfide or mitochondria-targeted donor molecules inhibits the hyperglycemia-induced ROS production and endothelial dysfunction at low concentrations [35, 94]. Long-term administration of H2 S is effective against diabetic nephropathy and retinopathy in animal models [159, 160].

glycemic control provides limited protection against the cardiovascular events, and adjunct therapy is necessary to reduce the risk of complications. Previous efforts found aldose reductase (a key enzyme of the polyol pathway) to serve as potential drug target and found that inhibition of aldose reductase may prevent the pathological changes that occur in response to high sorbitol levels [141]. The results of the clinical trials were less impressive than expected from preclinical studies, still the first aldose reductase inhibitor has been marketed in Asia for

Following the discovery that mitochondrial superoxide is responsible for the induction of all ROS-producing pathways in hyperglycemic endothelial cells, an intense search began for mitochondrial drug targets and inhibitors of mitochondrial superoxide generation [26, 83, 84]. Mitochondria-specific targeting moieties have been developed and linked to antioxidants or superoxide dismutase (SOD) mimetics [143, 144]. The majority of these molecules use the triphenylphosphonium (TPP) targeting group and attain 100- to 500-fold accumulation in the mitochondria [145, 146]. Both the mitochondria-targeted ubiquinol (MitoQ) and piperidine nitroxide TEMPO (mitoTEMPO) proved beneficial in diabetes models [147–149]. Overexpression of MnSOD was found to protect against diabetic retinopathy in a transgenic

While no protein target has been identified, various phenotypic screens were performed, which allowed the target-agnostic discovery of potential drug candidates [38, 151]. In cellbased models, paroxetine emerged as a mitochondrial free radical scavenger and glucocorticoid steroids as UCP2 inducers in hyperglycemic endothelial cells [35, 38]. In another screen, which also used multiple assay variables as output and examined the OXPHOS-associated gene expression, the antihelmintic drug mebendazole and the Chinese herbal medicine deoxysappanone B emerged as inhibitors of mitochondrial ROS production [151]. The protective effect of all of these drugs might be a direct consequence of the normalization of the

The mitochondrial antioxidant effect of select microtubular drugs, which was independently confirmed by the previous two studies, might be related to their mitochondrial fusion promoting effect, since excessive mitochondrial fission and fragmentation occur in hyperglycemia [38, 151]. To identify compounds that promote mitochondrial fusion, a separate phenotypic screening program was conducted [152]. This study identified hydrazone M1, a small molecule that restore the mitochondrial network in cells but its efficacy has not been tested in diabetes [152, 153]. Also, compounds that block the mitochondrial fission are expected to improve the mitochondrial function and reduce the mitochondrial ROS production in hyperglycemia, but these inhibitors (mitochondrial division inhibitor-1 (mdivi-1), dynasore, P110 and 15-oxospiramilactone) have not been tested in diabetes

In another effort to identify compounds that reduce the mitochondrial ROS production but do not interfere with energy production, isolated mitochondria were used for high throughput screening and hit compounds were selected based on a dual output of ROS production and respiration rate [156, 157]. CN-POBS (N-cyclohexyl-4-(4-nitrophenoxy)benzenesulfonamide)

model, but long-term delivery of MnSOD is unresolved in humans [150].

mitochondrial membrane potential or free radical scavenging.

[154, 155].

the treatment of diabetes complications [142].

196 Endothelial Dysfunction - Old Concepts and New Challenges

Finally, many of the currently used anti-diabetic medications also possess mitochondrial targets and they might be partly responsible for their beneficial effects on endothelial cells and the vasculature. The biguanide metformin apart from activating AMPK also acts as a mild and transient inhibitor of respiratory complex I [161]. The sulfonylurea glibenclamide inhibits the mitochondrial ATP-sensitive potassium channel (mitoKATP), decreases the mitochondrial membrane potential and ROS production and increases the respiration rate [162, 163]. Thiazolidinediones (TZDs) also possess a specific mitochondrial target (mTOT, mitochondrial target of TZDs), which comprise a recently identified protein complex. TZDs bind to a pyruvate carrier complex in the mitochondria and modulate the pyruvate entry into the mitochondria that may explain their antioxidant effect [164]. The discovery of these novel mitochondrial targets is expected to promote target-based drug discovery efforts and may provide new compounds in the upcoming years.
