**4. Changes in acetylcholine pathways**

20-hydroxy-eicosatrinoic acid (20-HETE), which is a vasoconstrictor. Cyclooxygenase (COX) is an enzyme existing in two isoformes, COX-1 and COX-2, involved in the synthesis of prostanoid from arachidonic acid (AA). The resulting prostanoids act in contradiction, causing vasodilation (prostaglandin D2, prostaglandin E2, and prostacyclin I2) and vasoconstriction (prostaglandin F2α and thromboxane A2). Hypoxia activates the COX pathway, where mostly prostacyclin, PGI2, is generated. It diffuses into the smooth muscle cell in which it activates the enzyme adenylate cyclase and increases the amount of cyclic adenosine monophosphate (cAMP). cAMP promotes the opening of several types of potassium channels, resulting in hyperpolarization of the smooth muscle membrane with consequent vasodilation [33]. Lipoxygenase is an enzyme that from AA generates 12- and 15-hydroxy eicosatrienoic

Streptozocin-induced diabetes in rats (a model for type 1 diabetes mellitus) reduces the levels of protective EETs, and the reduced EET levels lead to exacerbation of stroke [35]. Tsai et al. showed impaired endothelium-dependent vasodilation of coronary arterioles caused by reduced CYP activity and EET production due to increased glucose-induced superoxide levels in coronary endothelial cells [36]. EETs might constitute a key link between insulin resistance and endothelial dysfunction [37]. Endothelial dysfunction in diabetes could also be related to the release of vasoconstrictor mediators, e.g., increased production of 20-HETE leading to activation of ROS through an NAD(P)H-dependent pathway. Diabetes alters CYP expression and 20-HETE formation, leading to upregulation of CYP4A isoforms and to elevated levels of 20-HETE [37]. Li et al. also suggested contribution of 20-HETE to endothelial dysfunction in diabetes and other insulin-resistant conditions showing the attenuation of diabetes-induced vascular dysfunction by using the 20-HETE inhibitor HET0016 [38]. Insulin-stimulated vasodilation mediated by the IRS-1/PI3K/AKT/eNOS pathway can be impaired by 20-HETE [39]. Issan et al. associated dysfunction of circulating endothelial progenitor cells and angiogenic capacity with increased levels of CYP-derived 20-HETE in diabetic patients with cardiac ischemia [39]. P450 4A metabolite 20-HETE by vascular tissue is directly dependent on the con-

centration of oxygen within the normal physiological range of blood and tissue PO<sup>2</sup>

require only very low PO<sup>2</sup>

is known that various arachidonic acid metabolites (prostaglandins, EETs, HETEs) and NO are of utmost importance in the mediation of vascular reactions to vasodilators and vasoconstrictors [41–46], including hypoxia and hyperoxia stimuli [46]. In conditions of reduced blood flow, the use of HBO can significantly increase tissue oxygenation. Although all P450 enzymes require molecular oxygen, the majority of them (such as those found in the liver)

that hyperbaric oxygen increases vascular sensitivity to EETs, instead of significantly increasing EET synthesis [3]. Our studies also show that HBO is a highly effective treatment for stroke even in the presence of long-term untreated diabetes, by inhibition of 20-HETE production [47]. Unfirer et al.'s study showed changes in the dilatation mechanisms in diabetic rats under the influence of hyperbaric oxygenation. It has been shown that hyperbaric oxygenation causes activation of the CYP450 epoxygenase pathway and increased EET production in diabetic animals exposed to HBO [13]. Furthermore, Kibel et al. showed a changed relaxation response to ANG-(1–7) influenced by HBO in healthy and diabetic animals, where they also linked to a changed mechanism and improved relaxation after HBO with CYP450 activation and EET synthesis [3, 11]. HBO was shown to increase relaxation responses to ANG-(1–7) in rat aortic rings of diabetic animals, and this effect was eliminated with the addition of an EET

levels for normal activity. Results from our previous study suggest

[40]. It

acids (HETEs) as the major active metabolites in the endothelial cell [29, 34].

90 Hyperbaric Oxygen Treatment in Research and Clinical Practice - Mechanisms of Action in Focus

In the literature, there are a lot of studies on animal models of diabetes mellitus that confirmed impaired mechanisms of vasodilation and vasoconstriction. Streptozotocin-induced diabetes mellitus in rats demonstrates attenuated vasodilation response to acetylcholine [56, 57]. Experiments on healthy mouse coronary arteries demonstrate that vasodilation to acetylcholine is accomplished 50% by NO and 50% by EDHF. In spontaneously diabetic mouse type II (db/db), that ratio is 81% to production of EDHF [12].

Unfirer et al. [13] first investigated mechanisms of vasorelaxation in diabetic animal models after HBO exposure. Thoracic aortal rings from SD rats were used to evaluate vasorelaxation responses to acetylcholine after preconstruction with noradrenalin. With NG-nitro-L-arginine methyl ester (L-NAME)-(NOS inhibitor), indomethacin-(COX inhibitor), and N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide (MS-PPOH)-(CYP 450-epoxygenase inhibitor), they investigated which pathway is involved in enhanced vasorelaxation responses in diabetic and healthy rats after HBO exposure. HBO exposure protocol was performed in therapeutic range [58]. DM duration of 6 weeks did not change vasorelaxation response in diabetic group, and after application of inhibitors, results showed that the NO pathway is dominant in macrocirculation. In the diabetic and healthy groups, after HBO exposure, there was partial inhibition of vasorelaxation after NOS inhibition, which indicates that other pathways were included in vasorelaxation mechanisms. MS-PPOH partially blocked vasorelaxation in both HBO groups, which indicates that HBO changes vasorelaxation mechanisms to alternative pathways—enhanced production or sensitivity to EETs. Indomethacin did not inhibit vasorelaxation in any group, so COX pathway did not have influence. These findings were verified with upregulation of eNOS and COX-1 enzymes in the diabetic HBO group and higher protein expression of CYP450-4A1/A2/A3 in both HBO groups when compared with their respective controls. Also in this study, there was not oxidative stress caused by HBO because thiobarbituric acid-reactive substances (TBARSs) were elevated in DM group but were normal in the healthy HBO group. This difference between studies is probably a result of different experimental protocols (intermittent hyperbaric oxygenation—2 hours, 4 days at 2.0 atm abs vs. 90 minutes, 7 days at 2.4 atm abs in Matsunami study [59]).

hydrogen peroxide (H<sup>2</sup>

O2

regulation of antioxidant enzyme activity of tissues [74].

been attributed to the enhanced ROS production induced by the HBO<sup>2</sup>

**6. Inflammation**

tive therapeutic effects of HBO<sup>2</sup>

), singlet oxygen (O<sup>2</sup>

ide dismutases, catalases, glutathione S-transferases, and thioredoxins [69].

rous acids (HOCl) [67]. Sometimes when ROSs break the upper concentration limit of cellular antioxidant defense system capacity, based on high ROS intracellular concentration or low cellular antioxidant defense system, oxidative stress will show up and manifest with nucleic acids, proteins, and lipids damage, leading to carcinogenesis, neurodegenerative disorders, atherosclerosis, diabetes, and aging [68]. Under normal physiological conditions, ROS and the peroxidized molecules are neutralized by a powerful antioxidant system involving superox-

In diabetes and hyperglycemia in general, NADPH oxidase represents the principal source of ROS production in different organs [67]. The most acceptable thesis is that oxidative stress, as a main result of HBO, is a major trigger of most of its effects, but the exact mechanisms are not completely clear. It could be confusing to understand different consequences of HBO depending on protocol type that was used. For example, the duration of exposure, the used oxygen pressure, the subject species, and the underlying disease are factors that may play a role in changes of blood pressure levels [70], and changes of specific oxidative parameters depend on lapsed time after exposure or on the number of repeated exposures (analyzing rat lung tissue) [71, 72]. Although increased superoxide dismutase and glutathione peroxidase activity and increased thiobarbituric acid-reactive substance levels are documented, after some hyperbaric protocols, there is no change in aforementioned enzyme concentrations in red blood cells. On the other hand, a significant induction of heat shock protein HSP70 in lymphocytes after even a single HBO2 treatment was noted—this might be due to activation of compensatory mechanisms by HBO2

After hyperbaric treatment with high oxygen concentration, an increased ROS production is noticed, but paradoxically, HBO induces an antioxidant environment in plasma by increasing the plasma catalase activity. Different studies have documented increases in the total plasma antioxidant capacity determined after a session with HBO [73]. The therapeutic use of HBO can give positive results by activation of ROS resulting in increased perfusion, reduced edema, decreased inflammatory cytokines, increased fibroblast proliferation, increased collagen production, and angiogenesis promotion. Finally, increase of ROS may improve the

Pathological effects of DM on the vascular wall include enhanced ROS production and endothelial activation leading to inflammation, atherogenesis, and vascular dysfunction, which further results in clinical impairment of the micro- and macrocirculation. Interestingly, posi-

Numerous studies on experimental DM animal models revealed ongoing vascular inflammation under diabetic/hyperglycemic conditions, characterized by (a) increased proinflammatory cytokine levels, including interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α); (b) endothelial activation followed by increased expression of vascular cellular adhesion molecule-1 (VCAM-1);

, such as antioxidative and anti-inflammatory effects, have

treatment [1].

, high-energy form of oxygen), and hypochlo-

http://dx.doi.org/10.5772/intechopen.76569

Mechanisms of HBO-Induced Vascular Functional Changes in Diabetic Animal Models

[70].

93

Same authors investigate HBO effect on microcirculation (middle cerebral arteries) in diabetic animal model, 6-week duration of DM. Preliminary results shown impaired vasodilation response in diabetic rats and restored vasodilation after HBO exposure. Using inhibitors such as indomethacin (COX), NG-monomethyl-L-arginine (L-NMMA) (NOS), and clotrimazole (nonselective CYP 450 inhibitor), they notice shift in vasodilation mechanisms from mainly NO pathway toward two other pathways COX/CYP 450 because in both HBO groups, L-NMMA did not blocked vasodilation to acetylcholine. Further investigation is necessary [60].

In normal condition, vasodilation response to hypoxia is made by activating cyclooxygenase (COX) and production of prostacyclin (PGI2) [61]. There is evidence that CYP 450-epoxigenase enzyme in minor part causes vasodilation in healthy vessels [62]. Experiments on middle cerebral arteries (MCAs) of 6 weeks diabetic rats that underwent HBO exposure were used to evaluate the effect of HBO in acute hypoxia. They used COX inhibitor indomethacin and selective CYP 450 epoxygenase inhibitor MS-PPOH. COX inhibition partially preserved vasodilation in HBO groups, and eliminated vasodilation in response to hypoxia in the presence of MS-PPOH in both HBO groups suggests that HBO activates CYP450-epoxigenase in MCAs of healthy and DM rats and shifts vasodilation mechanisms in response to acute hypoxia [63].
