Mythical metrics and methods: Needed paradigm shift in disease recognition and therapy.

Current medicine is practiced in an organ-based, function-appraised manner with less attention paid to the tissue characteristics of the appraised organs. The fundamentals of this paradigm have been the product of an oversimplified and often layman-based perceptions of the studied organ over the years. These perceptions drove the current definitions of normality and abnormality, parameters used in the diagnosis of the disease, goals of treatment and studied outcomes. Despite the explosive advancement in technology that could have potentially changed our 'upstream' thinking, practitioners remain captives of these old beliefs and have streamlined current technology in a 'downstream' fashion; in the form of goal-directed protocols, and engineering systems that would study their implementations. As a result, diseases continue to evolve, become more resistant to therapy, late to diagnose, and with a persistent worsening of outcomes. With a primarily focus on the heart and from an anesthesiologist prospective, we challenge the fundamentals of the current paradigm from an 'upstream' prospective. We challenge the current 'territorial' definitions of the organs studied, the current terminology of some diseases, the parameters used in their diagnosis, the diagnostic modalities used and their goals of treatment. We illustrate some examples when the current collective 'myth' meets the 'reality' in an acute care setting, further clarifying the limitations of the current paradigm. We also, provide a theoretical hypothesis of what we believe to be a potential substitute of the current paradigm. Our theory redefines disease from an organ-based functional phenomenon to a structural-based tissue phenomenon, calling for an integrative and holistic approach of tissue assessment rather than a discrete approach that may potentially obscure the interaction of non-appraised organs. We also believe in redirecting technology in an upstream direction to better redefine and early detect diseases rather than submitting to generationally inherited beliefs. Whereas we have started some of our research on our proposed paradigm, our theoretical framework remains to be thought-provoking, and hypothesis-generating at the present time.

Opsin 3 and 4 mediate light-induced pulmonary vasorelaxation that is potentiated by G protein-coupled receptor kinase 2 inhibition.

We recently demonstrated that blue light induces vasorelaxation in the systemic mouse circulation, a phenomenon mediated by the nonvisual G protein-coupled receptor melanopsin (Opsin 4; Opn4). Here we tested the hypothesis that nonvisual opsins mediate photorelaxation in the pulmonary circulation. We discovered Opsin 3 (Opn3), Opn4, and G protein-coupled receptor kinase 2 (GRK2) in rat pulmonary arteries (PAs) and in pulmonary arterial smooth muscle cells (PASMCs), where the opsins interact directly with GRK2, as demonstrated with a proximity ligation assay. Light elicited an intensity-dependent relaxation of PAs preconstricted with phenylephrine (PE), with a maximum response between 400 and 460 nm (blue light). Wavelength-specific photorelaxation was attenuated in PAs from Opn4-/- mice and further reduced following shRNA-mediated knockdown of Opn3. Inhibition of GRK2 amplified the response and prevented physiological desensitization to repeated light exposure. Blue light also prevented PE-induced constriction in isolated PAs, decreased basal tone, ablated PE-induced single-cell contraction of PASMCs, and reversed PE-induced depolarization in PASMCs when GRK2 was inhibited. The photorelaxation response was modulated by soluble guanylyl cyclase but not by protein kinase G or nitric oxide. Most importantly, blue light induced significant vasorelaxation of PAs from rats with chronic pulmonary hypertension and effectively lowered pulmonary arterial pressure in isolated intact perfused rat lungs subjected to acute hypoxia. These findings show that functional Opn3 and Opn4 in PAs represent an endogenous "optogenetic system" that mediates photorelaxation in the pulmonary vasculature. Phototherapy in conjunction with GRK2 inhibition could therefore provide an alternative treatment strategy for pulmonary vasoconstrictive disorders.

Arginase Inhibition Reverses Endothelial Dysfunction, Pulmonary Hypertension, and Vascular Stiffness in Transgenic Sickle Cell Mice.

BACKGROUND: In sickle cell disease (SCD), hemolysis results in the release and activation of arginase, an enzyme that reciprocally regulates nitric oxide (NO) synthase activity and thus, NO production. Simply supplementing the common substrate L-arginine, however, fails to improve NO bioavailability. In this study, we tested the hypothesis that arginase inhibition would improve NO bioavailability and thereby attenuate systemic and pulmonary vascular endothelial dysfunction in transgenic mice with SCD. METHODS: We studied 5-month-old transgenic sickle cell (SC) mice and age matched wild-type (WT) controls. SC mice were treated with the arginase inhibitor, 2(S)-amino-6-boronohexanoic acid (ABH; approximately 400 µg/d) for 4 weeks or left untreated. RESULTS: Vascular arginase activity was significantly higher at baseline in untreated SC mice compared to WT controls (SC versus WT, 346 ± 69.3 vs 69 ± 17.3 pmol urea/mg protein/minute; P = 0.0043; n = 4-5 animals per group). Treatment with ABH may significantly decrease arginase activity to levels near WT controls (SC + ABH 125.2 ± 17.3 pmol urea/mg protein/minute; P = 0.0213). Aortic strips from untreated SC mice showed decreased NO and increased reactive oxygen species (ROS) production (NO: fluorescence rate 0.76 ± 0.14 vs 1.34 ± 0.17 RFU/s; P = 0.0005 and ROS: fluorescence rate 3.96 ± 1.70 vs 1.63 ± 1.20 RFU/s, P = 0.0039; n = 3- animals per group). SC animals treated with ABH for 4 weeks demonstrated NO (fluorescence rate: 1.16 ± 0.16) and ROS (fluorescence rate: 2.02 ± 0.45) levels comparable with age-matched WT controls (n = 3- animals per group). The maximal endothelial-dependent vasorelaxation response to acetylcholine was impaired in aortic rings from SC mice compared with WT (57.7% ± 8.4% vs 80.3% ± 11.0%; P = 0.02; n = 6 animals per group). The endothelial-independent response was not different between groups. In SC mice, the right ventricular cardiac output index and end-systolic elastance were similar (4.60 ± 0.51 vs 2.9 ± 0.85 mL/min/100 g and 0.89 ± 0.48 vs 0.58 ± 0.11 mm Hg/µL), whereas the pulmonary vascular resistance index and right ventricular end-systolic pressure were greater (2.9 ± 0.28 vs 5.5 ± 2.0 mm Hg × min/µL/100 g and 18.9 ± 1.1 vs 23.1 ± 4.0 mm Hg; n = 8 animals per group). Pulse wave velocity (a measure of arterial stiffness) was greater in SC mice compared with WT (3.74 ± 0.54 vs 3.25 ± 0.21 m/s; n = 20 animals per group), arginase inhibition for 4 weeks significantly reduced the vascular SC phenotype to one similar to WT animals (P = 0.0009). CONCLUSIONS: Arginase inhibition improves NO bioavailability and thereby attenuates systemic and pulmonary vascular endothelial dysfunction in transgenic mice with SCD. Therefore, arginase is a potential therapeutic target in the treatment of cardiovascular dysfunction in SCD.

2,3-Diphosphoglycerate Concentrations in Autologous Salvaged Versus Stored Red Blood Cells and in Surgical Patients After Transfusion.

BACKGROUND: Stored red blood cells (RBCs) are deficient in 2,3-diphosphoglycerate (2,3-DPG), but it is unclear how autologous salvaged blood (ASB) compares with stored blood and how rapidly 2,3-DPG levels return to normal after transfusion. Therefore, we compared levels of 2,3-DPG in stored versus ASB RBCs and in patients' blood after transfusion. METHODS: Twenty-four patients undergoing multilevel spine fusion surgery were enrolled. We measured 2,3-DPG and the oxyhemoglobin dissociation curve (P50) in samples taken from the ASB and stored blood bags before transfusion and in blood samples drawn from patients before and after transfusion. RESULTS: The mean storage duration for stored RBCs was 24 ± 8 days. Compared with fresh RBCs, stored RBCs had decreased 2,3-DPG levels (by approximately 90%; P < 0.0001) and a decreased P50 (by approximately 30%; P < 0.0001). However, ASB RBCs did not exhibit these changes. The mean 2,3-DPG concentration decreased by approximately 20% (P < 0.05) in postoperative blood sampled from patients who received 1 to 3 stored RBC units and by approximately 30% (P < 0.01) in those who received ≥4 stored RBC units. 2,3-DPG was unchanged in patients who received no stored blood or ASB alone. After surgery, 2,3-DPG levels recovered gradually over 3 postoperative days in patients who received stored RBCs. CONCLUSIONS: Stored RBCs, but not ASB RBCs, have decreased levels of 2,3-DPG and a left-shift in the oxyhemoglobin dissociation curve. Postoperatively, 2,3-DPG levels remain below preoperative baseline levels for up to 3 postoperative days in patients who receive stored RBCs but are unchanged in those who receive only ASB RBCs.

Impaired red blood cell deformability after transfusion of stored allogeneic blood but not autologous salvaged blood in cardiac surgery patients.

BACKGROUND: Both cardiopulmonary bypass (CPB) and red blood cell (RBC) storage are associated with detrimental changes in RBC structure and function that may adversely affect tissue oxygen delivery. We tested the hypothesis that in cardiac surgery patients, RBC deformability and aggregation are minimally affected by CPB with autologous salvaged blood alone but are negatively affected by the addition of stored allogeneic blood. METHODS: In this prospective cohort study, 32 patients undergoing cardiac surgery with CPB were divided into 3 groups by transfusion status: autologous salvaged RBCs alone (Auto; n = 12), autologous salvaged RBCs + minimal (<5 units) stored allogeneic RBCs (Auto+Allo min; n = 10), and autologous salvaged RBCs + moderate (≥5 units) stored allogeneic RBCs (Auto+Allo mod; n = 10). Ektacytometry was used to measure RBC elongation index (deformability) and critical shear stress (aggregation) before, during, and for 3 days after surgery. RESULTS: In the Auto group, RBC elongation index did not change significantly from the preoperative baseline. In the Auto+Allo min group, mean elongation index decreased from 32.31 ± 0.02 (baseline) to 30.47 ± 0.02 (nadir on postoperative day 1) (P = 0.003, representing a 6% change). In the Auto+Allo mod group, mean elongation index decreased from 32.7 ± 0.02 (baseline) to 28.14 ± 0.01 (nadir on postoperative day 1) (P = 0.0001, representing a 14% change). Deformability then dose-dependently recovered toward baseline over the first 3 postoperative days. Changes in aggregation were unrelated to transfusion (no difference among groups). For the 3 groups combined, mean critical shear stress decreased from 359 ± 174 mPa to 170 ± 141 mPa (P = 0.01, representing a 54% change), with the nadir at the end of surgery and returned to baseline by postoperative day 1. CONCLUSIONS: In cardiac surgery patients, transfusion with stored allogeneic RBCs, but not autologous salvaged RBCs, is associated with a decrease in RBC cell membrane deformability that is dose-dependent and may persist beyond 3 postoperative days. These findings suggest that autologous salvaged RBCs may be of higher quality than stored RBCs, since the latter are subject to the so-called storage lesions.

Inhaled hydrogen sulfide improves graft function in an experimental model of lung transplantation.

OBJECTIVES: Ischemia/reperfusion injury (IRI) is a common complication of lung transplantation (LTx). Hydrogen sulfide (H(2)S) is a novel agent previously shown to slow metabolism and scavenge reactive oxygen species, potentially mitigating IRI. We hypothesized that pretreatment with inhaled H(2)S would improve graft function in an ex vivo model of LTx. METHODS: Rabbits (n = 10) were ventilated for 2 h prior to heart-lung bloc procurement. The treatment group (n = 5) inhaled room air (21% O(2)) supplemented with 150 ppm H(2)S while the control group (n = 5) inhaled room air alone. Both groups were gradually cooled to 34°C. All heart-lung blocs were then recovered and cold-stored in low-potassium dextran solution for 18 h. Following storage, the blocs were reperfused with donor rabbit blood in an ex vivo apparatus. Serial clinical parameters were assessed and serial tissue biochemistry was examined. RESULTS: Prior to heart-lung bloc procurement, rabbits pretreated with H(2)S exhibited similar oxygenation (P = 0.1), ventilation (P = 0.7), and heart rate (P = 0.5); however, treated rabbits exhibited consistently higher mean arterial blood pressures (P = 0.01). During reperfusion, lungs pretreated with H(2)S had better oxygenation (P < 0.01) and ventilation (P = 0.02), as well as lower pulmonary artery pressures (P < 0.01). Reactive oxygen species levels were lower in treated lungs during reperfusion (P = 0.01). Additionally, prior to reperfusion, treated lungs demonstrated more preserved mitochondrial cytochrome c oxidase activity (P = 0.01). CONCLUSIONS: To our knowledge, this study represents the first reported therapeutic use of inhaled H(2)S in an experimental model of LTx. After prolonged ischemia, lungs pretreated with inhaled H(2)S exhibited improved graft function during reperfusion. Donor pretreatment with inhaled H(2)S represents a potentially novel adjunct to conventional preservation techniques and merits further exploration.

Alagebrium in combination with exercise ameliorates age-associated ventricular and vascular stiffness.

Advanced glycation end-products (AGEs) initiate cellular inflammation and contribute to cardiovascular disease in the elderly. AGE can be inhibited by Alagebrium (ALT), an AGE cross-link breaker. Moreover, the beneficial effects of exercise on aging are well recognized. Thus, we investigated the effects of ALT and exercise (Ex) on cardiovascular function in a rat aging model. Compared to young (Y) rats, in sedentary old (O) rats, end-systolic elastance (Ees) decreased (0.9±0.2 vs 1.7±0.4mmHg/µL, P<0.05), dP/dt(max) was attenuated (6054±685 vs 9540±939mmHg/s, P<0.05), ventricular compliance (end-diastolic pressure-volume relationship (EDPVR)) was impaired (1.4±0.2 vs 0.5±0.4mmHg/µL, P<0.05) and diastolic relaxation time (tau) was prolonged (21±3 vs 14±2ms, P<0.05). In old rats, combined ALT+Ex (4weeks) increased dP/dt(max) and Ees (8945±665 vs 6054±685mmHg/s, and 1.5±0.2 vs 0.9±0.2 respectively, O with ALT+Ex vs O, P<0.05 for both). Diastolic function (exponential power of EDPVR and tau) was also substantially improved by treatment with Alt+Ex in old rats (0.4±0.1 vs 0.9±0.2 and 16±2 vs 21±3ms, respectively, O with ALT+EX vs O, P<0.05 for both). Pulse wave velocity (PWV) was increased in old rats (7.0±0.7 vs 3.8±0.3ms, O vs Y, P<0.01). Both ALT and Ex alone decreased PWV in old rats but the combination decreased PWV to levels observed in young (4.6±0.5 vs 3.8±0.3ms, O with ALT+Ex vs Y, NS). These results suggest that prevention of the formation of new AGEs (with exercise) and breakdown of already formed AGEs (with ALT) may represent a therapeutic strategy for age-related ventricular and vascular stiffness.

Oral atorvastatin therapy restores cutaneous microvascular function by decreasing arginase activity in hypercholesterolaemic humans.

Elevated low-density lipoproteins (LDLs) are associated with vascular dysfunction evident in the cutaneous microvasculature. We hypothesized that uncoupled endothelial nitric oxide synthase (NOS3) through upregulated arginase contributes to cutaneous microvascular dysfunction in hyperocholesterolaemic (HC) humans and that a statin intervention would decrease arginase activity. Five microdialysis fibres were placed in the skin of nine normocholesterolaemic (NC: LDL level 95±4 mg dl⁻¹) and nine hypercholesterolaemic (HC: LDL: 177±6 mg dl⁻¹) men and women before and after 3 months of systemic atrovastatin. Sites served as control, NOS inhibited, arginase inhibited, L-arginine supplemented and arginase inhibited plus L-arginine supplemented. Skin blood flow was measured while local skin heating (42°C) induced NO-dependent vasodilatation. L-NAME was infused after the established plateau in all sites to quantify NO-dependent vasodilatation. Data were normalized to maximum cutaneous vascular conductance (CVC(max)). Skin samples were obtained to measure total arginase activity and arginase I and arginase II protein. Vasodilatation was reduced in hyperocholesterolaemic subjects (HC: 76±2 vs. NC: 94±3%CVC(max), P < 0.001) as was NO-dependent vasodilatation (HC: 43±5 vs. NC: 62±4%CVC(max), P < 0.001). The plateau and NO-dependent vasodilatation were augmented in HC with arginase inhibition (92±2, 67±2%CVC(max), P < 0.001), L-arginine (93±2, 71±5%CVC(max), P < 0.001) and combined treatments (94±4, 65±5%CVC(max), P < 0.001) but not in NC. After statin intervention (LDL: 98±5 mg dl⁻¹) there was no longer a difference between control sites (88±4, 61±5%CVC(max)) and localized microdialysis treatment sites (all P > 0.05). Arginase activity and protein were increased in HC skin (P < 0.05 vs. NC) and activity decreased with atrovastatin treatment (P < 0.05). Reduced NOS3 substrate availability through upregulated arginase contributes to cutaneous microvascular dysfunction in hyperocholesterolaemic humans, which is corrected with atorvastatin therapy.

Neuronal nitric oxide synthase negatively regulates xanthine oxidoreductase inhibition of cardiac excitation-contraction coupling.

Although interactions between superoxide (O(2)(.-)) and nitric oxide underlie many physiologic and pathophysiologic processes, regulation of this crosstalk at the enzymatic level is poorly understood. Here, we demonstrate that xanthine oxidoreductase (XOR), a prototypic superoxide O(2)(.-) -producing enzyme, and neuronal nitric oxide synthase (NOS1) coimmunoprecipitate and colocalize in the sarcoplasmic reticulum of cardiac myocytes. Deficiency of NOS1 (but not endothelial NOS, NOS3) leads to profound increases in XOR-mediated O(2)(.-) production, which in turn depresses myocardial excitation-contraction coupling in a manner reversible by XOR inhibition with allopurinol. These data demonstrate a unique interaction between a nitric oxide and an O(2)(.-) -generating enzyme that accounts for crosstalk between these signaling pathways; these findings demonstrate a direct antioxidant mechanism for NOS1 and have pathophysiologic implications for the growing number of disease states in which increased XOR activity plays a role.