Phone: (706) 721-3384
Fax: (706) 721-2347

Email: wcaldwel@mail.mcg.edu

Office: CB-3622

Grant Support as Principal Investigator | Current Projects | Lab | Selected Publications |

Our laboratory is currently involved with several research projects:

1. Role of arginase in vascular endothelial dysfunction. L-arginase is metabolized by NOS to produce NO and L-citrulline. L-citrulline can be recycled back to L-arginine by argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL), which are co-localized in the caveolar area with eNOS and CAT1. L-arginine is also substrate for arginase, which catalyzes conversion of L-arginine to ornithine and urea (Figure 1). There are 2 arginase isoforms. Arginase I is a cytosolic enzyme that constitutes a majority of total body arginase activity. It is strongly expressed in the liver and is central to the urea cycle. The other form, arginase II, is mainly a mitochondrial enzyme particularly evident in the kidney, but also present in other cells. Arginase I activates the ornithine decarboxylase (ODC) pathway, producing polyamines which are important in cellular growth and migration and can contribute to cellular hypertrophy and hyperplasia. Arginase II plays a role in the production of proline, a critical component of collagen, through the ornithine aminotransferase (OAT)/pyrroline-5-carboxylate reductase (P5CR) pathway. Competition between NOS and arginase for L-arg within the cell to produce either NO or ornithine/urea is quite feasible given their individual enzymatic properties. Although the affinity of L-arginine is much higher for NOS (Km ~ 6 µM) than for arginase (Km ~ 5 mM), the maximum activity (Vmax) for arginase is greater than 1000 times that for NOS, indicating similar rates of substrate utilization at physiological L-arg levels.


Figure 1. Major enzymes in L-arginine metabolic pathway (OAT: ornithine amino transferase, OCT:. ornithine carbomyl transferase, CP: carbomyl phosphate, ASS: argininosuccinate synthase, ASL: argininosuccinate lyase, ODC: ornithine decarboxylase, NOS: NO synthase.

We hypothesize that diabetes and high glucose cause vascular dysfunction by activation of the small G protein RhoA and Rho kinase and subsequently activation of the arginase /ornithine pathway. Decreased L-arginine availability to eNOS uncouples the enzyme to form superoxide rather than NO which reduces vasodilation. Accelerated activity of the arginase /ornithine pathway induces perivascular fibrosis, hypertrophy, leading to vascular stiffening. This model is being examined using a combination of molecular, pharmacological and biochemical approaches to test the following hypotheses for experiments in diabetic animals, (including transgenic mice lacking the two isoforms of arginase) cells and blood from diabetic patients and cultured cells. 1. Define the molecular mechanisms by which diabetes activates arginase. 2. Test whether diabetes causes impaired EC-dependent vasorelaxation by activating arginase. 3. Test whether diabetes causes vascular fibrosis, hypertrophy and stiffening by activating arginase. 4. Test whether diabetes activates the arginase/ornithine pathway by activation of RhoA/ROCK.

2. Correction of cardiac dysfunction in diabetes by statins. Our overall aim is to determine the extent and means by which statins and L-arginine can protect the heart against diabetes-induced injury. Endothelial cell (EC) dysfunction, as indicated by impaired endothelium-dependent vasodilation, is a prominent feature in the vascular complications of diabetes and has been noted in a variety of species and organ beds in models of type I (insulin-dependent) diabetes. In animal models, dysfunction has been reported in many vascular beds including that of the heart. Clinical studies have also demonstrated impaired vasodilation in patients with type I diabetes. Coronary vascular dysfunction and increased susceptibly to ischemic myocardial injury are also evident in humans with diabetes. Statins, HMG CoA reductase inhibitors, are thought to prevent or reverse endothelial dysfunction through their pleiotropic effects - reduction in cholesterol and other lipids, reduction in reactive oxygen species (ROS) and other mediators of inflammation, as well as enhancement of physiological NO production. Statins lower ROS levels and reduce diabetic complications by a number of mechanisms, including NADPH oxidase activation and increasing NO production. Statins have been shown to promote angiogenesis and induce the mobilization of progenitor stem cells to angiogenic sites and to protect against infarction of brain and heart in animal models of ischemia. We are investigating mechanisms by which statins maintain or return cardiac, vascular and EC function to normal in diabetes. These studies are being performed in rats with streptozotocin-induced diabetes and in EC treated with high glucose levels. We hypothesize that statins and/or L-arginine or its precursor, L-citrulline exert their vascular and organ protective effect by reducing ROS and inflammation, enhancing L-arginine-availability and endothelial NOS function.

Sources of coronary vascular ROS include uncoupled nitric oxide synthase (NOS) and NADPH oxidase. In order to evaluate oxidative stress levels in the coronary arteries of the diabetic hearts and identify potential sources of ROS formation, we performed DHE imaging of fresh frozen sections of the cardiac ventricular septum of 8 week rats. Under identical reaction conditions, the DHE signal was much more intense within and around the coronary arteries of the diabetic rats than the controls. This increase in DHE staining was blocked by treatment with either L-NAME (NOS inhibitor, 3 mM) or apocynin (NADPH oxidase inhibitor, 30 µM), indicating that sources of superoxide production in diabetic vessels include both NOS and NADPH oxidase (Figure 2). Specificity of the reaction for superoxide was demonstrated by complete blockade of the signal by superoxide dismutase (SOD).

3. Substrate precursor of nitric oxide synthase. Increasing L-arginine levels or availability at the site of NOS activity can enhance NO production and prevent the production of O2.- by NOS. Elevation of extracellular L-arginine concentration to levels which allow entry of L-arginine into endothelial cells by passive diffusion or low affinity/high capacity transport is a means of providing adequate substrate. However, relatively large daily oral doses (~50mg/kg) appear to be required for this effect. A draw-back to administering L-arginine orally to elevate plasma levels of arginine is that a large portion of the L-arginine passing through the G.I. tract and the liver is catabolized by arginase I to ornithine. Furthermore, elevated levels of circulating L-arginine can induce arginase in the vasculature, kidney and macrophage which increases the rate of L-arginine catabolism. Thus, doses of L-arginine necessary to produce desired L-arginine blood levels have to be determined with this considerable catabolic loss in mind. Importantly, L-arginine is synthesized by conversion of L-citrulline to L-argininosuccinate (AS) by AS synthase (ASS) and its conversion to L-arginine by AS lyase (ASL). Both AS and ASS are present in the caveolae region of endothelial cells. Citrulline is also the product of NOS's action on L-arginine. Citrulline is thus a readily available and rapidly converted source of L-arginine. L-citrulline is not a substrate for arginase; infact, it is an inhibitor of arginase. Thus, small doses of citrulline raise arginine levels much more than arginine itself. A recent human study reported that oral administration of the same dose of either l-citrulline or L-arginine resulted in a 227% or 90% peak increase (4 hr) in plasma L-arginine levels, respectively. Furthermore, the area under the arginine plasma concentration - time curve was 3.5 fold larger for citrulline and the elevation in arginine levels was more enduring following citrulline administration. Thus, systemic administration of citrulline, the precursor of L-arginine, appears to be a more efficient way to elevate extracellular levels of L-arginine. We are examining the ability of supplemental and arginine to enhance the hypotensive response (NO production) to acetylcholine and the effects of these treatments on arginase activity in the liver, kidney and vasculature. Nitroglycerin (GTN) tolerance involves reactive oxygen species

L-arginine metabolism in diabetes-induced coronary dysfunction. PI (30% effort) NIH 1 RO1 HL70215 (2002-2012). Our goals have been to determine: 1) The effects of oxidant exposure and diabetes on cellular L-arginine transport. 2) The effects of limiting L-arginine on cellular production of NO, superoxide anion and peroxynitrite. 3) Whether oxidant exposure alters L-Arginine transport activity by altering membrane potential. We also are determing the effects of disease models of oxidative stress - diabetes and hyperhomocyteinemia - on L-arginine transport function and arginase activity.

Additionally, we hypothesize that diabetes and high glucose cause vascular dysfunction by activation of the small G protein RhoA and Rho kinase and subsequently activation of the arginase/ornithine pathway. To test this hypothesis we are:
1. Defining the molecular mechanisms by which diabetes activates arginase,
2. testing whether diabetes causes impaired EC-dependent vasorelaxation by activating arginase,
3. determining whether diabetes causes vascular fibrosis, hypertrophy and stiffening by activating arginase, and
4. testing whether diabetes activates the arginase/ornithine pathway by activation of RhoA/ROCK.

Cellular Mechanisms of Retinal Angiogenesis 2003-2012. 2R01 EY-11766, PI (15% effort). Based on our previous work and preliminary data, our global hypothesis is that NADPH oxidase-induced activation of the arginase pathway has a key role in causing retinal vascular dysfunction and inducing pathological angiogenesis and fibrosis/gliosis during retinopathy. Our specific aims are as follows:
1. Determine the role of NADPH oxidase-derived ROS in increasing arginase expression/activity and uncoupling NOS during retinopathy.
2. Define the molecular mechanisms of arginase activation during retinopathy.
3. Test whether diabetes causes retinal vascular dysfunction by activating arginase.
4. Test whether retinal ischemia causes pathological angiogenesis and fibrosis/gliosis by increasing arginase.

Blood Retinal Barrier Changes in Retinopathy 2001-2011. NIH EY 04618 Co-Investigator (10% effort). The major long term goal is to determine mechanisms which alter blood-retinal barrier function in retinal disease. The genesis of reactive oxygen species appears to be germinally involved in these processes and vascular dysfunction. Their role in diabetes is being examined.

1. Role of arginase, bioavailability of L-arginine, and reactive oxygen species in cardiac and vascular dysfunctions.
2. Actions and mechanisms of action of statins in cardiovascular complications of diabetes.
3. Involvement of reactive oxygen species in the vascular tolerance to nitroglycerin-type agents .

Maritza J. Romero, M.D., D.Sc., Assistant Research Scientist

Jennifer Iddings, B.S. - Research Assistant

Alia Shatanawi, B.D.S - Predoctoral Fellow

Surabhi Chandra, Ph.D. - Postdoctoral Fellow

Haroldo A. Toque, Ph.D. - Postdoctoral Fellow

Caldwell RB, Zhang W, Romero MJ, Caldwell RW. Does excess arginase activity play a role in retinopathy? Brain Research Bulletin. In press - April 2009.

Zhang W, Baban B, Rojas M, Tofigh S, Virmani SK, Patel C, Behzadian MA, Romero M, Caldwell RW, Caldwell RB. Arginase activity mediates retinal inflammation in endotoxin-induced uveitis. American Journal of Pathology, In Press - April 2009.

Zhang W, Rojas M, Lilly B, Tsai NT, Lemtalsi T, Liou GI, Caldwell RW, Caldwell RB. NAD(P)H oxidase regulates CCL2 production during retinal inflammation. Invest Ophthalmol Vis Sci. 2009 Feb 21. [Epub ahead of print]

Romero M, Platt D, Tawfik H, Labazi M, El-Remessy AB, Bartoli M, Caldwell RB, Caldwell RW. Diabetes-induced coronary vascular dysfunction involves increased arginase activity. Circulation Research, 2008, 102; 95-102.

Penn JS, Madan A, Caldwell RB, Bartoli M, Caldwell RW, Hartnett ME., Vascular endothelial growth Factor in eye disease. Prog Retin Eye Res. 2008, (4): 331-71.

Al-Shabrawey, M., Bartoli, M., El-Remessy, A.B., Ma, G., Matragoon, S., Lemtalsi, T., Caldwell, R.W., Caldwell, R.B. Role of NADPH oxidase and STAT3 in statin-mediated protection against diabetic retinopathy. Invest. Ophthalmol. Vis. Sci. Mar 31, 49(7);3231-8, 2008.

Yu Q, Nguyen T, Ogbi M, Caldwell R.W. and Johnson J.A. Differential loss of cytochrome c oxidase (CO) subunits in ischemia reperfusion injury: exacerbation of COI subunit loss by zPKC inhibition. Am. J. Physiol. (Heart/Circ.), 294 (6):H2637-45, 2008.

Guo, D., Nguyen, T., Ogbi, M., Tawfik, H., Ma, G., Yu, Q., Caldwell, RW, Johnson, J. Epsilon protein kinase C co-immunoprecipitates with cytochrome oxidase subunit IV and is associated with improved cytochrome c oxidase activity and cardio-protection. Am. J. Physiol. (Heart/Circ.) 293(4):H2219-30, 2007.

Jin, L., Caldwell, R.B., Li-Masters, T. and Caldwell, R.W. Homocysteine induces endothelial dysfunction via inhibition of arginine transport. J. Physiol. and Pharmacol. 58(2): 191-206, 2007.

Romero, M.J., Platt, D.H., Caldwell, R.B., Caldwell, R.W. Therapeutic use of citrulline in cardiovascular disease. Cardiovascular Drug Reviews 24(3-4): 275-290, 2006.

Tawfik, H.E., El-Remessy, A.B., Matragoon, S., Ma, G., Caldwell, R.B., Caldwell, R.W. Simvastatin improves diabetes-induced Coronary Endothelial Dysfunction. J. Pharmacol. Exp. Therap. 319(1): 386-95, 2006.

Ma, G., Al-Shabrawey, M., Johnson, J., Datar R., Caldwell, R.B., Caldwell, R.W. Protection against myocardial ischemia/reperfusion injury by short term diabetes: role of VEGF-induced angiogenesis and activation of cell survival signaling. Naunyn-Schmiedeberg's Archives of Pharmacology. 373(6):415-27, 2006.

Banes-Berceli, AK, Shaw, S, Ma, G, Brands, M, Eaton, DC, Stern, DM, Fulton, D., Caldwell, R.W., Marrero, M.B. Effect of simvastatin on high glucose-and angiotensin II-Induced activation of the JAK/STAT pathway in mesangial cells. Am. J. Physiol. Renal Physiol. 291(1): F116-21, 2006.

Tawfik, A, Jin, L. , Banes-Berceli, A.M., Caldwell, R.B., Ogbi S., Shirley A., Barber D., Catravas J.D., Stern D.M., Fulton D., Caldwell, R.W., Marrero, M. Hyperglycemia and reactive oxygen species mediate apoptosis in aortic endothelial cells through Janus kinase 2. Vascular Pharmacology, 43(5): 320-326, 2005.

Al-Shabrawey, M., Bartoli, M., El-Remessy, A.B., Platt, D.H., Matragoon, S. M.Ali Behzadian, M.A., Caldwell, R.W. and Caldwell, R.B. Inhibition of NAD(P)H oxidase activity blocks VEGF over-expression and neovascularization during ischemic retinopathy. Am. J. Pathology. 167(2):599-607, 2005.

Caldwell RB, Bartoli M, Ali Behzadian M, El-Remessy A, Al-Shabrawey M, Platt D, Liou GI, Caldwell, R.W. Vascular Endothelial Growth Factor and Diabetic Retinopathy: Role of Oxidative Stress. Current Drug Targets. 6(4):51-524, 2005.

Ergul A, Schultz Johansen J, Stromhaug C, Harris AK, Hutchinson J, Tawfik A, Rahimi A, Rhim E, Wells B, Caldwell RW, Anstadt MP. Vascular dysfunction of venous bypass conduits is mediated by reactive oxygen species in diabetes: Role of endothelin-1. J. Pharmacol. Exptl. Therap. 2004 Dec. 17. [Epub ahead of print]

Kaesemeyer, W.H., Jin, L. Caldwell, R.B., Caldwell, R.W. Drug-Induced Endothelial Cell Dysfunction. In "Nitric Oxide and its Biomedical Significance" Ed: George Stefano, Medical Science International, Ltd. 2004.

Abou-Mohamed, G., J.A. Johnson, L.Jin, A.B. El-Remessy, K. Do, W.H. Kaesemeyer, R.B. Caldwell, and R.W. Caldwell. Roles of Superoxide, Peroxynitrite and Protein Kinase C in the Development of Tolerance to Nitroglycerin), J. Pharmacol. Exptl. Therap. 308(1): 289-299, 2004.

Caldwell, R.B., Bartoli, M., Behzadian, M.A., El-Remessy, A.E., El-Shabrawey, M., Platt, D.H., Caldwell, R.W. Vascular endothelial growth factor and diabetic retinopathy: pathophysiological mechanisms and treatment perspectives. Diabetes Metab. Res. Rev. 19(6): 442-55, 2003.

El-Remessy,A. Khalil, I. Abou-Mohamed, G. Matragoon, S. Caldwell, R.B., Caldwell,R.W., and Liou, G.I. Neuroprotective effect of delta 9-tetrahydrocannabinol and cannabidiol in NMDA-induced retinal neurotoxicity: involvement of peroxynitrite. Amer. J. Path. 163(5):1997-2008, 2003.

Abou-Mohamed, G., A. El-Marakby, J.D. Catravas, G.O. Carrier, R.W. Caldwell, and R.E. White. Estradiol relaxes rat aorta via endothelium-dependent and -independent mechanisms. Pharmacology 66:20-26, 2003.

El-Remessy, A.B., Behzadian, M.A., Abou-Mohamed, G., Franklin, T., Caldwell, R.W., and Caldwell, R.B. Experimental diabetes causes breakdown of the blood-retina barrier by a mechanism involving tyrosine nitration and increases in expression of vascular endothelial growth factor and urokinase plasminogen activator receptor. Am. J. Pathol. 162(6);1995-2004. 2003.

El-Remessy, A.B., Abou-Mohamed, G., Caldwell, R.W., and Caldwell, R.B. High glucose-induced tyrosine nitration in endothelial cells: role of eNOS uncoupling and aldose reductase activation. Invest. Opthalmol. Vis. Sci. 44(7): 3135- 43, 2003.

Parker, J.O., Parker, J.D., Caldwell, R. W., Farrel, B., Kaesemeyer, W.H. The effect of supplemental L-arginine on tolerance development during continuous transdermal nitroglycerin therapy. J. Am. Coll. Card. 39(7): 1199-1203, 2002.

Jin, L., Abou-Mohamed, G., R.B. Caldwell and Caldwell R.W. Endothelial cell dysfunction in a model of oxidative stress. Medical Science Monitor. 7(4):585-591, 2001.

Abou-Mohamed, G., Huang, J., Oldham, C.D, Taylor, T.A., Jin, L., Caldwell, R.B., May, S.W. and Caldwell, R.W. Vascular and endothelial actions of inhibitors of peptide amidation. J. Cardiov. Pharmacol. 35:871-880, 2000.

Kaesemeyer, W.H., Ogonowski, A.A., Jin, L., Caldwell, R.B. and Caldwell, R.W. Endothelial nitric oxide synthase is a site of superoxide synthesis in endothelial cells treated with glyceryl trinitrate. Brit. J. Pharmacol. 131:1019-1023, 2000.

Abou-Mohamed, G., Kaesemeyer, W.H., Caldwell, R.B. and Caldwell, R.W. Role of L-arginine in the development and reversal of tolerance to nitroglycerin. Brit. J. Pharmacol. 130:211-218, 2000.

Ogonowski, A.A., Kaesemeyer, W.H., Jin, L., Ganapathy, V., Leibach, F.H. and Caldwell, R.W. Effect of nitric oxide donors and synthase agonists on endothelial cell uptake of L-arginine and superoxide anion production. Am. J. Physiol. (Cell Physiol). 278 (1); C136-C143, 2000.

Caldwell RB, Zhang W, Romero MJ, Caldwell RW. Nitric oxide synthase, oxidative stress and retinal vascular dysfunction: Emerging role of arginase. Brain Research Bulletin. In press - July, 2009.

Zhang W, Baban B, Rojas M, Tofigh S, Virmani SK, Patel C, Behzadian MA, Romero M, Caldwell RW, Caldwell RB. Arginase activity mediates retinal inflammation in endotoxin-induced uveitis. American Journal of Pathology, In Press - April 2009. [Epub ahead of print, July 2009].

Zhang W, Rojas M, Lilly B, Tsai NT, Lemtalsi T, Liou GI, Caldwell RW, Caldwell RB. NAD(P)H oxidase-dependent regulation of CCL2 production during retinal inflammation. Invest Ophthalmol Vis Sci. 50(6):3033-40, June 2009.