A Potential Role for Excess Tissue Iron in Development of Cardiovascular Delayed Effects of Acute Radiation Exposure.

Murine hematopoietic-acute radiation syndrome (H-ARS) survivors of total body radiation (TBI) have a significant loss of heart vessel endothelial cells, along with increased tissue iron, as early as 4 mo post-TBI. The goal of the current study was to determine the possible role for excess tissue iron in the loss of coronary artery endothelial cells. Experiments used the H-ARS mouse model with gamma radiation exposure of 853 cGy (LD50/30) and time points from 1 to 12 wk post-TBI. Serum iron was elevated at 1 wk post-TBI, peaked at 2 wk post-TBI, and returned to non-irradiated control values by 4 wk post-TBI. A similar trend was seen for transferrin saturation, and both results correlated inversely with red blood cell number. Perls' Prussian Blue staining, used to detect iron deposition in heart tissue sections, showed myocardial iron was present as early as 2 wk following irradiation. Pretreatment of mice with the iron chelator deferiprone decreased tissue iron but not serum iron at 2 wk. Coronary artery endothelial cell density was significantly decreased as early as 2 wk vs. non-irradiated controls (P<0.05), and the reduced density persisted to 12 wk after irradiation. Deferiprone treatment of irradiated mice prevented the decrease in endothelial cell density at 2 and 4 wk post-TBI compared to irradiated, non-treated mice (P<0.03). Taken together, the results suggest excess tissue iron contributes to endothelial cell loss early following TBI and may be a significant event impacting the development of delayed effects of acute radiation exposure.

Development of a Model of the Acute and Delayed Effects of High Dose Radiation Exposure in Jackson Diversity Outbred Mice; Comparison to Inbred C57BL/6 Mice.

Development of medical countermeasures against radiation relies on robust animal models for efficacy testing. Mouse models have advantages over larger species due to economics, ease of conducting aging studies, existence of historical databases, and research tools allowing for sophisticated mechanistic studies. However, the radiation dose-response relationship of inbred strains is inherently steep and sensitive to experimental variables, and inbred models have been criticized for lacking genetic diversity. Jackson Diversity Outbred (JDO) mice are the most genetically diverse strain available, developed by the Collaborative Cross Consortium using eight founder strains, and may represent a more accurate model of humans than inbred strains. Herein, models of the Hematopoietic-Acute Radiation Syndrome and the Delayed Effects of Acute Radiation Exposure were developed in JDO mice and compared to inbred C57BL/6. The dose response relationship curve in JDO mice mirrored the more shallow curves of primates and humans, characteristic of genetic diversity. JDO mice were more radioresistant than C57BL/6 and differed in sensitivity to antibiotic countermeasures. The model was validated with pegylated-G-CSF, which provided significantly enhanced 30-d survival and accelerated blood recovery. Long-term JDO survivors exhibited increased recovery of blood cells and functional bone marrow hematopoietic progenitors compared to C57BL/6. While JDO hematopoietic stem cells declined more in number, they maintained a greater degree of quiescence compared to C57BL/6, which is essential for maintaining function. These JDO radiation models offer many of the advantages of small animals with the genetic diversity of large animals, providing an attractive alternative to currently available radiation animal models.

Cardiac and Renal Delayed Effects of Acute Radiation Exposure: Organ Differences in Vasculopathy, Inflammation, Senescence and Oxidative Balance.

We have previously shown significant pathology in the heart and kidney of murine hematopoietic-acute radiation syndrome (H-ARS) survivors of 8.7-9.0 Gy total-body irradiation (TBI). The goal of this study was to determine temporal relationships in the development of vasculopathy and the progression of renal and cardiovascular delayed effects of acute radiation exposure (DEARE) at TBI doses less than 9 Gy and to elucidate the potential roles of senescence, inflammation and oxidative stress. Our results show significant loss of endothelial cells in coronary arteries by 4 months post-TBI (8.53 or 8.72 Gy of gamma radiation). This loss precedes renal dysfunction and interstitial fibrosis and progresses to abnormalities in the arterial media and adventitia and loss of coronary arterioles. Major differences in radiation-induced pathobiology exist between the heart and kidney in terms of vasculopathy progression and also in indices of inflammation, senescence and oxidative imbalance. The results of this work suggest a need for different medical countermeasures for multiple targets in different organs and at various times after acute radiation injury to prevent the progression of DEARE.

Novel method to assess arterial insufficiency in rodent hind limb.

BACKGROUND: Lack of techniques to assess maximal blood flow capacity thwarts the use of rodent models of arterial insufficiency to evaluate therapies for intermittent claudication. We evaluated femoral vein outflow (VO) in combination with stimulated muscle contraction as a potential method to assess functional hind limb arterial reserve and therapeutic efficacy in a rodent model of subcritical limb ischemia. MATERIALS AND METHODS: VO was measured with perivascular flow probes at rest and during stimulated calf muscle contraction in young, healthy rats (Wistar Kyoto, WKY; lean Zucker rats, LZR) and rats with cardiovascular risk factors (spontaneously hypertensive [SHR]; obese Zucker rats [OZR]) with acute and/or chronic femoral arterial occlusion. Therapeutic efficacy was assessed by administration of Ramipril or Losartan to SHR after femoral artery excision. RESULTS: VO measurement in WKY demonstrated the utility of this method to assess hind limb perfusion at rest and during calf muscle contraction. Although application to diseased models (OZR and SHR) demonstrated normal resting perfusion compared with contralateral limbs, a significant reduction in reserve capacity was uncovered with muscle stimulation. Administration of Ramipril and Losartan demonstrated significant improvement in functional arterial reserve. CONCLUSIONS: The results demonstrate that this novel method to assess distal limb perfusion in small rodents with subcritical limb ischemia is sufficient to unmask perfusion deficits not apparent at rest, detect impaired compensation in diseased animal models with risk factors, and assess therapeutic efficacy. The approach provides a significant advance in methods to investigate potential mechanisms and novel therapies for subcritical limb ischemia in preclinical rodent models.

Delayed Effects of Acute Radiation Exposure in a Murine Model of the H-ARS: Multiple-Organ Injury Consequent to <10 Gy Total Body Irradiation.

The threat of radiation exposure from warfare or radiation accidents raises the need for appropriate animal models to study the acute and chronic effects of high dose rate radiation exposure. The goal of this study was to assess the late development of fibrosis in multiple organs (kidney, heart, and lung) in survivors of the C57BL/6 mouse model of the hematopoietic-acute radiation syndrome (H-ARS). Separate groups of mice for histological and functional studies were exposed to a single uniform total body dose between 8.53 and 8.72 Gy of gamma radiation from a Cs radiation source and studied 1-21 mo later. Blood urea nitrogen levels were elevated significantly in the irradiated mice at 9 and 21 mo (from ∼22 to 34 ± 3.8 and 69 ± 6.0 mg dL, p < 0.01 vs. non-irradiated controls) and correlated with glomerosclerosis (29 ± 1.8% vs. 64 ± 9.7% of total glomeruli, p < 0.01 vs. non-irradiated controls). Glomerular tubularization and hypertrophy and tubular atrophy were also observed at 21 mo post-total body irradiation (TBI). An increase in interstitial, perivascular, pericardial and peribronchial fibrosis/collagen deposition was observed from ∼9-21 mo post-TBI in kidney, heart, and lung of irradiated mice relative to age-matched controls. Echocardiography suggested decreased ventricular volumes with a compensatory increase in the left ventricular ejection fraction. The results indicate that significant delayed effects of acute radiation exposure occur in kidney, heart, and lung in survivors of the murine H-ARS TBI model, which mirrors pathology detected in larger species and humans at higher radiation doses focused on specific organs.

Impaired compensation to femoral artery ligation in diet-induced obese mice is primarily mediated via suppression of collateral growth by Nox2 and p47phox.

The present study was undertaken to establish the role of NADPH oxidase (Nox) in impaired vascular compensation to arterial occlusion that occurs in the presence of risk factors associated with oxidative stress. Diet-induced obese (DIO) mice characterized by multiple comorbidities including diabetes and hyperlipidemia were used as a preclinical model. Arterial occlusion was induced by distal femoral artery ligation in lean and DIO mice. Proximal collateral arteries were identified as the site of major (∼70%) vascular resistance to calf perfusion by distal arterial pressures, which decreased from ∼80 to ∼30 mmHg with ligation in both lean and DIO mice. Two weeks after ligation, significant vascular compensation occurred in lean but not DIO mice as evidenced by increased perfusion (147 ± 48% vs. 49 ± 29%) and collateral diameter (151 ± 30% vs. 44 ± 17%). Vascular mRNA expression of p22(phox), Nox2, Nox4, and p47(phox) were all increased in DIO mice. Treatment of DIO mice with either apocynin or Nox2ds-tat or with whole body ablation of either Nox2 or p47(phox) ameliorated the impairment in both collateral growth and hindlimb perfusion. Multiparametric flow cytometry analysis demonstrated elevated levels of circulating monocytes in DIO mice without impaired mobilization and demargination after femoral artery ligation. These results establish collateral resistance as the major limitation to calf perfusion in this preclinical model, demonstrate than monocyte mobilization and demarginatin is not suppressed, implicate Nox2-p47(phox) interactions in the impairment of vascular compensation to arterial occlusion in DIO mice, and suggest that selective Nox component suppression/inhibition may be effective as either primary or adjuvant therapy for claudicants.

Nox2 and p47(phox) modulate compensatory growth of primary collateral arteries.

The role of NADPH oxidase (Nox) in both the promotion and impairment of compensatory collateral growth remains controversial because the specific Nox and reactive oxygen species involved are unclear. The aim of this study was to identify the primary Nox and reactive oxygen species associated with early stage compensatory collateral growth in young, healthy animals. Ligation of the feed arteries that form primary collateral pathways in rat mesentery and mouse hindlimb was used to assess the role of Nox during collateral growth. Changes in mesenteric collateral artery Nox mRNA expression determined by real-time PCR at 1, 3, and 7 days relative to same-animal control arteries suggested a role for Nox subunits Nox2 and p47(phox). Administration of apocynin or Nox2ds-tat suppressed collateral growth in both rat and mouse models, suggesting the Nox2/p47(phox) interaction was involved. Functional significance of p47(phox) expression was assessed by evaluation of collateral growth in rats administered p47(phox) small interfering RNA and in p47(phox-/-) mice. Diameter measurements of collateral mesenteric and gracilis arteries at 7 and 14 days, respectively, indicated no significant collateral growth compared with control rats or C57BL/6 mice. Chronic polyethylene glycol-conjugated catalase administration significantly suppressed collateral development in rats and mice, implying a requirement for H2O2. Taken together, these results suggest that Nox2, modulated at least in part by p47(phox), mediates early stage compensatory collateral development via a process dependent upon peroxide generation. These results have important implications for the use of antioxidants and the development of therapies for peripheral arterial disease.

Molecular basis for impaired collateral artery growth in the spontaneously hypertensive rat: insight from microarray analysis.

Analysis of global gene expression in mesenteric control and collateral arteries was used to investigate potential molecules, pathways, and mechanisms responsible for impaired collateral growth in the Spontaneously Hypertensive Rat (SHR). A fundamental difference was observed in overall gene expression pattern in SHR versus Wistar Kyoto (WKY) collaterals; only 6% of genes altered in collaterals were similar between rat strains. Ingenuity® Pathway Analysis (IPA) identified major differences between WKY and SHR in networks and biological functions related to cell growth and proliferation and gene expression. In SHR control arteries, several mechano-sensitive and redox-dependent transcription regulators were downregulated including JUN (-5.2×, P = 0.02), EGR1 (-4.1×, P = 0.01), and NFĸB1 (-1.95×, P = 0.04). Predicted binding sites for NFĸB and AP-1 were present in genes altered in WKY but not SHR collaterals. Immunostaining showed increased NFĸB nuclear translocation in collateral arteries of WKY and apocynin-treated SHR, but not in untreated SHR. siRNA for the p65 subunit suppressed collateral growth in WKY, confirming a functional role of NFkB. Canonical pathways identified by IPA in WKY but not SHR included nitric oxide and renin-angiotensin system signaling. The angiotensin type 1 receptor (AGTR1) exhibited upregulation in WKY collaterals, but downregulation in SHR; pharmacological blockade of AGTR1 with losartan prevented collateral luminal expansion in WKY. Together, these results suggest that collateral growth impairment results from an abnormality in a fundamental regulatory mechanism that occurs at a level between signal transduction and gene transcription and implicate redox-dependent modulation of mechano-sensitive transcription factors such as NFĸB as a potential mechanism.

Marvels, mysteries, and misconceptions of vascular compensation to peripheral artery occlusion.

Peripheral arterial disease is a major health problem and there is a significant need to develop therapies to prevent its progression to claudication and critical limb ischemia. Promising results in rodent models of arterial occlusion have generally failed to predict clinical success and led to questions of their relevance. While sub-optimal models may have contributed to the lack of progress, we suggest that advancement has also been hindered by misconceptions of the human capacity for compensation and the specific vessels which are of primary importance. We present and summarize new and existing data from humans, Ossabaw miniature pigs, and rodents which provide compelling evidence that natural compensation to occlusion of a major artery (i) may completely restore perfusion, (ii) occurs in specific pre-existing small arteries, rather than the distal vasculature, via mechanisms involving flow-mediated dilation and remodeling (iii) is impaired by cardiovascular risk factors which suppress the flow-mediated mechanisms and (iv) can be restored by reversal of endothelial dysfunction. We propose that restoration of the capacity for flow-mediated dilation and remodeling in small arteries represents a largely unexplored potential therapeutic opportunity to enhance compensation for major arterial occlusion and prevent the progression to critical limb ischemia in the peripheral circulation.

Antioxidants reverse age-related collateral growth impairment.

Aging is a major risk factor for the development of cardiovascular diseases, including arterial occlusive disease. Oxidant stress increases with age, and may be a significant factor contributing to vascular dysfunction and disease. We have shown that aging and hypertension impair collateral growth, the natural compensatory response to arterial occlusive disease, and that antioxidants restore collateral growth in young hypertensive rats. The aim of this study was to test the hypothesis that oxidant stress mediates collateral growth impairment in nondiseased, aged rats. Ileal arteries were induced to become collaterals via ligation of adjacent arteries. Growth was assessed at 7 days by repeated in vivo measurements and comparison to same-animal control arteries. Collateral diameter enlargement did not occur in aged rats, but luminal expansion was stimulated by pretreatment with tempol. Co-administration of L-NAME with tempol prevented tempol-mediated collateral development. Expression of p22(phox) mRNA was increased in aged versus young rat arteries, suggesting NAD(P)H oxidase as a source of reactive oxygen species. Treatment with apocynin increased collateral growth capacity, whether administered prior to, or 7 days following, arterial ligation. The results suggest that antioxidant treatment may be useful in promoting collateral growth to compensate for age-related arterial occlusive disease.

Abnormal nitric oxide production in aged rat mesenteric arteries is mediated by NAD(P)H oxidase-derived peroxide.

Previous work in our laboratory showed increased basal periarterial nitric oxide (NO) and H2O2 concentrations in the spontaneously hypertensive rat, characterized by oxidant stress, as well as impaired flow-mediated NO production that was corrected by a reduction of periarterial H2O2. Aging is also associated with an increase in vascular reactive oxygen species and results in abnormal vascular function. The current study was designed to assess the role of H2O2 in regulating NO production during vascular aging. In vivo, real-time NO and H2O2 concentrations were measured by microelectrodes in mesenteric arteries of retired breeder (aged; 8-12 mo) and young (2 to 3 mo) Wistar-Kyoto rats under conditions of altered flow. The results in aged rats revealed elevated basal NO (1,611+/-286 vs. 793+/-112 nM, P<0.05) and H2O2 concentrations (16+/-2 vs. 9+/-1 microM, P<0.05) and a flow-mediated increase in H2O2 but not NO production. Pretreatment of aged rats with the antioxidant apocynin lowered both basal H2O2 (8+/-1 microM) and NO (760+/-102 nM) to young levels and restored flow-mediated NO production. Similar results were obtained with the NAD(P)H oxidase inhibitor gp91ds-tat. In addition, acute incubation with topical polyethylene-glycolated catalase lowered the baseline NO concentration and restored flow-mediated NO production. Taken together, the data indicate that elevated baseline and suppressed flow-mediated NO production in aged Wistar-Kyoto rats are mediated by NAD(P)H oxidase-derived H2O2.

Suppressed hindlimb perfusion in Rac2-/- and Nox2-/- mice does not result from impaired collateral growth.

While tissue perfusion and angiogenesis subsequent to acute femoral artery occlusion are suppressed in NADPH oxidase 2 (Nox2)-null (Nox2(-/-)) mice, studies have not established the role of Nox2 in collateral artery enlargement. Rac2 is a small GTPase that binds Nox2 and activates Nox2-based NAD(P)H oxidase but, unlike Nox2, is primarily restricted to bone marrow-derived cells. In this study, we used Rac2-null (Rac2(-/-)) and Nox2(-/-) mice with a novel method of identifying primary hindlimb collaterals to investigate the hypothesis that collateral growth requires these molecules. When initial experiments performed with femoral ligation demonstrated similar perfusion and collateral growth in Rac2(-/-) and wild-type C57BL/6J (BL6) mice, subsequent experiments were performed with a more severe ischemia model, femoral artery excision. After femoral excision, tissue perfusion was suppressed in Rac2(-/-) mice relative to BL6 mice. Histological assessment of ischemic injury including necrotic and regenerated muscle fibers and lipid and collagen deposition demonstrated greater injury in Rac2(-/-) mice. The diameters of primary collaterals identified during Microfil injection with intravital microscopy were enlarged to a similar extent in BL6 and Rac2(-/-) mice. Intimal cells in collateral cross sections were increased in number in both strains and were CD31 positive and CD45 negative. Circulating leukocytes and CD11b(+) cells were increased more in Rac2(-/-) than BL6 animals. Experiments performed in Nox2(-/-) mice to verify that the unexpected results related to collateral growth were not unique to Rac2(-/-) mice gave equivalent results. The data demonstrate that, subsequent to acute femoral artery excision, perfusion recovery is impaired in Rac2(-/-) and Nox2(-/-) mice but that collateral luminal expansion and intimal cell recruitment/proliferation are normal. These novel results indicate that collateral luminal expansion and intimal cell recruitment/proliferation are not mediated by Rac2 and Nox2.

NAD(P)H oxidase-derived peroxide mediates elevated basal and impaired flow-induced NO production in SHR mesenteric arteries in vivo.

Nitric oxide (NO) and reactive oxygen species (ROS) have fundamentally important roles in the regulation of vascular tone and remodeling. Although arterial disease and endothelial dysfunction alter NO and ROS levels to impact vasodilation and vascular structure, direct measurements of these reactive species under in vivo conditions with flow alterations are unavailable. In this study, in vivo measurements of NO and H2O2 were made on mesenteric arteries to determine whether antioxidant therapies could restore normal NO production in spontaneously hypertensive rats (SHR). Flow was altered from approximately 50-200% of control in anesthetized Wistar-Kyoto rats (WKY) and SHR by selective placement of microvascular clamps on adjacent arteries while NO and H2O2 were directly measured with microelectrodes. Relative to WKY, SHR had significantly increased baseline NO and H2O2 concentrations (2,572 +/- 241 vs. 1,059 +/- 160 nM, P < 0.01; and 26 +/- 7 vs. 7 +/- 1 microM, P < 0.05, respectively). With flow elevation, H2O2 but not NO increased in SHR; NO but not H2O2 was elevated in WKY. Apocynin and polyethylene-glycolated catalase decreased baseline SHR NO and H2O2 to WKY levels and restored flow-mediated NO production. Suppression of NAD(P)H oxidase with gp91ds-tat decreased SHR H2O2 to WKY levels. Addition of topical H2O2 to increase peroxide to the basal concentration measured in SHR elevated WKY NO to levels observed in SHR. The results support the hypothesis that increased vascular peroxide in SHR is primarily derived from NAD(P)H oxidase and increases NO concentration to levels that cannot be further elevated with increased flow. Short-term and even acute administration of antioxidants are able to restore normal flow-mediated NO signaling in young SHR.

Impact of genetic background and aging on mesenteric collateral growth capacity in Fischer 344, Brown Norway, and Fischer 344 x Brown Norway hybrid rats.

Available studies indicate that both genetic background and aging influence collateral growth capacity, but it is not known how their combination affects collateral growth. We evaluated collateral growth induced by ileal artery ligation in Fischer 344 (F344), Brown Norway (BN), and the first generation hybrid of F344 x BN (F1) rats available for aging research from the National Institute on Aging. Collateral growth was determined by paired diameter measurements in anesthetized rats immediately and 7 days postligation. In 3-mo-old rats, significant collateral growth occurred only in BN (35% +/- 11%, P < 0.001). The endothelial cell number in arterial cross sections was also determined, since this precedes shear-mediated luminal expansion. When compared with the same animal controls, the intimal cell number was increased only in BN rats (92% +/- 21%, P < 0.001). The increase in intimal cell number and the degree of collateral luminal expansion in BN rats was not affected by age from 3 to 24 mo. Immunohistochemical studies demonstrated that intimal cell proliferation was much greater in the collaterals of BN than of F1 rats. The remarkable difference between these three strains of rats used in aging research and the lack of an age-related impairment in the BN rats are novel observations. These rat strains mimic clinical observations of interindividual variation in collateral growth capacity and the impact of age on arteriogenesis and should be useful models to investigate the molecular mechanisms responsible for such differences.

Development of progressive aortic vasculopathy in a rat model of aging.

Recent studies have established that age is the major risk factor for vascular disease. Numerous aberrant changes occur in vascular structure and function during aging, and animal models are the primary means to determine the underlying mechanisms of age-mediated vascular pathology. The Fischer 344/Brown Norway F1 hybrid (F344xBN) rat thoracic aorta has been shown to display age-related pathology similar to what occurs in humans. This study utilized the F344xBN rat aorta and both morphometric and global gene expression analyses to identify appropriate time points to study vascular aging and to identify molecules associated with the development and progression of vascular pathology. In contrast to some previous studies that indicated age-related abrupt changes, a progressive increase in intimal and medial thickness, as well as smooth muscle cell-containing intimal protrusions, was observed in thoracic aorta. This structural vascular pathology was associated with a progressive, but nonlinear, increase in global differential gene expression. Gene products with altered mRNA and protein expression included inflammation-related molecules: specifically, the adhesion molecules ICAM-1 and VCAM-1 and the bone morphogenic proteins osteopontin and bone sialoprotein-1. Intimal-associated macrophages were found to increase significantly in number with age. Both systemic and tissue markers of oxidant stress, serum 8-isoprostane and 3-nitrotyrosine, respectively, were also found to increase during aging. The results demonstrate that major structural abnormalities and altered gene expression develop after 6 mo and that the progressive pathological development is associated with increased inflammation and oxidant stress.

Involvement of MMPs in the outward remodeling of collateral mesenteric arteries.

Persistent elevation in shear stress within conduit or resistance arteries causes structural luminal expansion, which serves to normalize shear stress while maintaining increased flow to the downstream vasculature. Although it is known that this adaptation involves cellular proliferation and remodeling of the extracellular matrix, the specific cellular events underlying these responses are poorly understood. Matrix metalloproteinases (MMPs) contribute to extensive remodeling of the extracellular matrix in conduit vessels and vein grafts exposed to high flow. However, involvement of MMPs in remodeling of small muscular collateral arteries, which are exposed to less severe increases in shear stress, has not been tested. We utilized an established model of outward remodeling in mesenteric collateral arteries to determine whether MMPs were upregulated during the remodeling response and to test whether MMP activity was required for luminal expansion. By 4 days, MMP-2 and membrane type 1 MMP (MT1-MMP), but not MMP-9, protein levels were significantly elevated in collateral arteries, as assessed by gelatin zymography and immunostaining. MMP-2 and MT1-MMP proteins, together with their respective transcriptional activators c-Jun and Egr-1 were localized predominantly to the smooth muscle layer of the collateral arteries. The general MMP inhibitor doxycycline prevented luminal expansion of collateral arteries but did not affect the endothelial cell proliferative or medial growth responses. In conclusion, this study provides evidence that MMP-2 and MT1-MMP are upregulated in collateral arteries exposed to elevated shear stress and that MMP activity is essential for the full remodeling response that leads to outward luminal expansion.

The role of the renin-angiotensin system and oxidative stress in spontaneously hypertensive rat mesenteric collateral growth impairment.

Recent clinical and animal studies have shown that collateral artery growth is impaired in the presence of vascular risk factors, including hypertension. Available evidence suggests that angiotensin-converting enzyme inhibitors (ACEI) promote collateral growth in both hypertensive humans and animals; however, the specific mechanisms are not established. This study evaluated the hypothesis that collateral growth impairment in hypertension is mediated by excess superoxide produced by NAD(P)H oxidase in response to stimulation of the ANG II type 1 receptor. After ileal artery ligation, mesenteric collateral growth did not occur in untreated, young, spontaneously hypertensive rats. Significant luminal expansion occurred in collaterals of spontaneously hypertensive rats treated with the superoxide dismutase mimetic tempol, the NAD(P)H oxidase inhibitor apocynin, and the ACEI captopril, but not ANG II type 1 (losartan) or type 2 (PD-123319) receptor blockers. The ACEI enalapril produced equivalent reduction of arterial pressure as captopril but did not promote luminal expansion. This suggests the effects of captopril on collateral growth might result from its antioxidant properties. RT-PCR demonstrated that ANG II type 1 receptor and angiotensinogen expression was reduced in collaterals of untreated rats. This local suppression of the renin angiotensin system provides a potential explanation for the lack of effect of enalapril and losartan on collateral growth. The results demonstrate the capability of antioxidant therapies, including captopril, to reverse impaired collateral artery growth and the novel finding that components of the local renin angiotensin system are naturally suppressed in collaterals.

Collateral growth in the peripheral circulation: a review.

Arterial occlusive diseases are a major cause of morbidity and death in the United States. The enlargement of pre-existing vessels, which bypass the site of arterial occlusion, provide a natural way for the body to compensate for such obstructions. Individuals differ in their capacity to develop collateral vessels. In recent years much attention has been focused upon therapy to promote collateral development, primarily using individual growth factors. Such studies have had mixed results. Persistent controversies exist regarding the initiating stimuli, the processes involved in enlargement, the specific vessels that should be targeted, and the most appropriate terminology. Consequently, it is now recognized that more research is needed to extend our knowledge of the complex process of collateral growth. This basic science review addresses five questions essential in understanding current problems in collateral growth research and the development of therapeutic interventions.

Impaired collateral artery development in spontaneously hypertensive rats.

OBJECTIVE: To determine whether collateral artery development is impaired in spontaneously hypertensive (SHR) relative to normotensive (WKY) rats. METHODS: Sequential mesenteric arteries were ligated to create a collateral pathway responsible for the perfusion of approximately 50 first-order arterioles. Collateral development was assessed by measurement of in vivo arterial diameter before and 1 week after ligation. Histological and morphometric measurements were made from cross-sectional preparations of these arteries to evaluate intimal and medial cell numbers and medial area. eNOS expression was evaluated with Western blotting. RESULTS: One week after arterial ligation, collateral diameter was increased more in WKY than SHR both absolutely (137 +/- 9.1 versus 99 +/- 8.6 microm) and relative to same-animal controls (38 +/- 5.5% versus 13 +/- 7.1%). At the time of model creation, blood flow was elevated to comparable levels in both WKY and SHR, and wall shear rate in the SHR collateral was greater than both the SHR control and WKY collateral arteries. Endothelial cell number in arterial cross-section was increased in collaterals by 80% in WKY and only 22% in the SHR. eNOS expression was increased in the WKY (128%) but not in the SHR collateral. CONCLUSIONS: For equivalent arterial occlusion, the data demonstrate that collateral development is suppressed in the SHR as indicated by luminal expansion. This impairment of luminal expansion is associated with a decreased endothelial proliferation and the lack of an increase in eNOS expression.