Christopher Kolz

Vascular Endothelial Growth Factor Is Required for Coronary Collateral Growth in the Rat

Background— The goal of this study was to determine whether the expression of vascular endothelial growth factor (VEGF) is critical for coronary collateral growth. Previous studies have provided an association between coronary collateral growth and VEGF, but none have allowed determination of a causal role. Methods and Results— We measured coronary collateral growth in rats subjected to repetitive episodes of myocardial ischemia (RI; one 40-second occlusion every 20 minutes for 2 hours 40 minutes, followed by 5 hours 20 minutes of rest, with this 8-hour cycle repeated 3 times per day for 10 days). Collateral growth was measured from blood flow (radioactive microspheres), visualization of arterial-arterial anastomoses (x-ray micro-CT), and maintenance of function during complete coronary occlusion in 3 groups of animals: sham (received instrumentation but no RI), experimental (subjected to RI), and anti–vascular endothelial growth factor (RI+anti-VEGF 0.6 mg/100 g per day) to block the endogenous actions of VEGF. In the 3 groups, native collateral flow (measurement for RI or sham protocol) averaged 0.2 to 0.3 mL · min−1 · g−1 of tissue. In the sham group, collateral flow did not increase during the protocol. Collateral flow in the control RI group increased by ≈6-fold to 1.63 mL · min−1 · g−1 tissue, but in the anti-VEGF group, collateral flow did not increase after the RI protocol (0.22 mL · min−1 · g−1). In acute experiments, collateral flow was unchanged during vasodilation with dipyridamole, indicating the increases in collateral flow are due to collateral growth and not vasodilation. X-ray micro-CT analysis revealed a 3-fold increase (versus sham group) in the number of arterial-arterial anastomoses per heart after RI, which was prevented by treatment with anti-VEGF. The growth of the collateral circulation was functional in the RI group because complete coronary occlusion did not induce any untoward effects on hemodynamics or arrhythmias. In the sham or anti-VEGF groups, coronary occlusion at the end of the protocol induced many arrhythmias and deterioration of function. Conclusions— From these results, we conclude that the expression of VEGF is critical to the growth of coronary collaterals.

The Mechanistic Basis for the Disparate Effects of Angiotensin II on Coronary Collateral Growth

Objective—We hypothesize that controversial effects of angiotensin II (Ang II) are attributable to its regulation of reactive oxygen species (ROS) and ROS-dependent signaling. Methods and Results—Coronary collateral growth (CCG) was stimulated in normal (WKY) and syndrome X (JCR) rats by transient/repetitive ischemia (RI). Blood flow was measured in the normal (NZ) and the collateral-dependent (CZ) zone. In WKY, RI increased CZ flow (0.84 mL/min/g), but RI+subpressor Ang II increased it more (1.24 mL/min/g). This was associated with transient p38 and sustained Akt activation. A hypertensive dose of Ang II decreased CZ flow (0.69 mL/min/g), which was associated with sustained p38 and transient Akt activation. AT1R blockade by candesartan abrogated CZ flow in WKY (0.58 mL/min/g), reduced myocardial superoxide, and blocked p38 and Akt activation. RI-induced CZ flow in JCR was significantly decreased compared with WKY (0.12 mL/min/g), associated with a large increase in superoxide and lack of p38 and Akt activation. CZ flow in JCR was partially restored by candesartan (0.45 mL/min/g), accompanied by reduction in superoxide and partial restoration of p38 and Akt activation. Conclusion—Ang II/AT1R blockade, at least in part, regulates CCG via generating optimal ROS amounts and activating redox-sensitive signaling.

Induction of Vascular Progenitor Cells From Endothelial Cells Stimulates Coronary Collateral Growth

Rationale: A well-developed coronary collateral circulation improves the morbidity and mortality of patients following an acute coronary occlusion. Although regenerative medicine has great potential in stimulating vascular growth in the heart, to date there have been mixed results, and the ideal cell type for this therapy has not been resolved. Objective: To generate induced vascular progenitor cells (iVPCs) from endothelial cells, which can differentiate into vascular smooth muscle cells (VSMCs) or endothelial cells (ECs), and test their capability to stimulate coronary collateral growth. Methods and Results: We reprogrammed rat ECs with the transcription factors Oct4, Klf4, Sox2, and c-Myc. A population of reprogrammed cells was derived that expressed pluripotent markers Oct4, SSEA-1, Rex1, and AP and hemangioblast markers CD133, Flk1, and c-kit. These cells were designated iVPCs because they remained committed to vascular lineage and could differentiate into vascular ECs and VSMCs in vitro. The iVPCs demonstrated better in vitro angiogenic potential (tube network on 2-dimensional culture, tube formation in growth factor reduced Matrigel) than native ECs. The risk of teratoma formation in iVPCs is also reduced in comparison with fully reprogrammed induced pluripotent stem cells (iPSCs). When iVPCs were implanted into myocardium, they engrafted into blood vessels and increased coronary collateral flow (microspheres) and improved cardiac function (echocardiography) better than iPSCs, mesenchymal stem cells, native ECs, and sham treatments. Conclusions: We conclude that iVPCs, generated by partially reprogramming ECs, are an ideal cell type for cell-based therapy designed to stimulate coronary collateral growth.

Stimulation of Coronary Collateral Growth by Granulocyte Stimulating Factor. Role of Reactive Oxygen Species

Objective—The purpose of this study was to determine whether G-CSF promotes coronary collateral growth (CCG) and decipher the mechanism for this stimulation. Methods and Results—In a rat model of repetitive episodic myocardial ischemia (RI, 40 seconds LAD occlusion every 20 minutes for 2 hours and 20 minutes, 3 times/d for 5 days) CCG was deduced from collateral-dependent flow (flow to LAD region during occlusion). After RI, G-CSF (100 &mgr;g/kg/d) increased CCG (P<0.01) (0.47±0.15) versus vehicle (0.14±0.06). Surprisingly, G-CSF treatment without RI increased CCG (0.57±0.18) equal to G-CSF+RI. We evaluated ROS by dihydroethidine (DHE) fluorescence (LV injection, 60 &mgr;g/kg, during two episodes of ischemia). DHE fluorescence was double in G-CSF+RI versus vehicle+RI (P<0.01), and even higher in G-CSF without RI (P<0.01). Interestingly, the DHE signal did not colocalize with myeloperoxidase (immunostaining, neutrophil marker) but appeared in cardiac myocytes. The study of isolated cardiac myocytes revealed the cytokine stimulates ROS which elicit production of angiogenic factors. Apocynin inhibited G-CSF effects both in vivo and in vitro. Conclusions—G-CSF stimulates ROS production directly in cardiomyocytes, which plays a pivotal role in triggering adaptations of the heart to ischemia including growth of the coronary collaterals.

Mitochondrial Oxidative Stress Corrupts Coronary Collateral Growth by Activating Adenosine Monophosphate Activated Kinase-α Signaling

Objective—Our goal was to determine the mechanism by which mitochondrial oxidative stress impairs collateral growth in the heart. Approach and Results—Rats were treated with rotenone (mitochondrial complex I inhibitor that increases reactive oxygen species production) or sham-treated with vehicle and subjected to repetitive ischemia protocol for 10 days to induce coronary collateral growth. In control rats, repetitive ischemia increased flow to the collateral-dependent zone; however, rotenone treatment prevented this increase suggesting that mitochondrial oxidative stress compromises coronary collateral growth. In addition, rotenone also attenuated mitochondrial complex I activity and led to excessive mitochondrial aggregation. To further understand the mechanistic pathway(s) involved, human coronary artery endothelial cells were treated with 50 ng/mL vascular endothelial growth factor, 1 µmol/L rotenone, and rotenone/vascular endothelial growth factor for 48 hours. Vascular endothelial growth factor induced robust tube formation; however, rotenone completely inhibited this effect (P<0.05 rotenone versus vascular endothelial growth factor treatment). Inhibition of tube formation by rotenone was also associated with significant increase in mitochondrial superoxide generation. Immunoblot analyses of human coronary artery endothelial cells with rotenone treatment showed significant activation of adenosine monophosphate activated kinase (AMPK)-&agr; and inhibition of mammalian target of rapamycin and p70 ribosomal S6 kinase. Activation of AMPK-&agr; suggested impairments in energy production, which was reflected by decrease in O2 consumption and bioenergetic reserve capacity of cultured cells. Knockdown of AMPK-&agr; (siRNA) also preserved tube formation during rotenone, suggesting the negative effects were mediated by the activation of AMPK-&agr;. Conversely, expression of a constitutively active AMPK-&agr; blocked tube formation. Conclusions—We conclude that activation of AMPK-&agr; during mitochondrial oxidative stress inhibits mammalian target of rapamycin signaling, which impairs phenotypic switching necessary for the growth of blood vessels.

Impaired coronary metabolic dilation in the metabolic syndrome is linked to mitochondrial dysfunction and mitochondrial DNA damage

Mitochondrial dysfunction in obesity and diabetes can be caused by excessive production of free radicals, which can damage mitochondrial DNA. Because mitochondrial DNA plays a key role in the production of ATP necessary for cardiac work, we hypothesized that mitochondrial dysfunction, induced by mitochondrial DNA damage, uncouples coronary blood flow from cardiac work. Myocardial blood flow (contrast echocardiography) was measured in Zucker lean (ZLN) and obese fatty (ZOF) rats during increased cardiac metabolism (product of heart rate and arterial pressure, i.v. norepinephrine). In ZLN increased metabolism augmented coronary blood flow, but in ZOF metabolic hyperemia was attenuated. Mitochondrial respiration was impaired and ROS production was greater in ZOF than ZLN. These were associated with mitochondrial DNA (mtDNA) damage in ZOF. To determine if coronary metabolic dilation, the hyperemic response induced by heightened cardiac metabolism, is linked to mitochondrial function we introduced recombinant proteins (intravenously or intraperitoneally) in ZLN and ZOF to fragment or repair mtDNA, respectively. Repair of mtDNA damage restored mitochondrial function and metabolic dilation, and reduced ROS production in ZOF; whereas induction of mtDNA damage in ZLN reduced mitochondrial function, increased ROS production, and attenuated metabolic dilation. Adequate metabolic dilation was also associated with the extracellular release of ADP, ATP, and H2O2 by cardiac myocytes; whereas myocytes from rats with impaired dilation released only H2O2. In conclusion, our results suggest that mitochondrial function plays a seminal role in connecting myocardial blood flow to metabolism, and integrity of mtDNA is central to this process.

Kv1.3 Channels Facilitate the Connection Between Metabolism and Blood Flow in the Heart

The connection between metabolism and flow in the heart, metabolic dilation, is essential for cardiac function. We recently found redox‐sensitive Kv1.5 channels play a role in coronary metabolic dilation; however, more than one ion channel likely plays a role in this process as animals null for these channels still showed limited coronary metabolic dilation. Accordingly, we examined the role of another Kv1 family channel, the energetically linked Kv1.3 channel, in coronary metabolic dilation. We measured myocardial blood flow (contrast echocardiography) during norepinephrine‐induced increases in cardiac work (heart rate x mean arterial pressure) in WT, WT mice given correolide (preferential Kv1.3 antagonist), and Kv1.3‐null mice (Kv1.3−/−). We also measured relaxation of isolated small arteries mounted in a myograph. During increased cardiac work, myocardial blood flow was attenuated in Kv1.3−/− and in correolide‐treated mice. In isolated vessels from Kv1.3−/− mice, relaxation to H2O2 was impaired (vs WT), but responses to adenosine and acetylcholine were equivalent to WT. Correolide reduced dilation to adenosine and acetylcholine in WT and Kv1.3−/−, but had no effect on H2O2‐dependent dilation in vessels from Kv1.3−/− mice. We conclude that Kv1.3 channels participate in the connection between myocardial blood flow and cardiac metabolism.