Elizabeth M. Wagner

Angiogenesis in the Mouse Lung

When pulmonary arterial blood flow is obstructed in all mammals studied, there is a compensatory growth of the bronchial vasculature. This angiogenesis normally occurs through a proliferation of the systemic circulation to the intraparenchymal airways. It is an important pathophysiological process, not only in pulmonary vascular disease, but also in lung cancer, because the blood flow that supplies primary lung tumors arises from the systemic circulation. In the mouse, however, the systemic blood vessels that supply the trachea and mainstem bronchi do not penetrate into the intraparenchymal airways, as they do in all other larger species. In this study, we attempted to generate a new functional bronchial circulation in the mouse by permanently obstructing 40% of the pulmonary circulation. We quantified the systemic blood flow to the lung with fluorescent microspheres for 3 months after left pulmonary artery ligation. Results demonstrated that a substantial systemic blood flow to the lung that can eventually supply up to 15% of the normal pulmonary flow can be generated beginning 5-6 days after ligation. These new angiogenic vessels do not arise from the extraparenchymal bronchial circulation. Rather they enter the lung directly via a totally new vasculature that develops between the visceral and parietal pleuras, supplied by several intercostal arteries. This unique model of angiogenesis occurs in the absence of any hypoxic stimulus and mimics the vascular source of many lung tumors.

mechanisms of Bronchoprotection by Anesthetic Induction Agents : Propofol versus Ketamine

BACKGROUND Propofol and ketamine have been purported to decrease bronchoconstriction during induction of anesthesia and intubation. Whether they act on airway smooth muscle or through neural reflexes has not been determined. We compared propofol and ketamine to attenuate the direct activation of airway smooth muscle by methacholine and limit neurally mediated bronchoconstriction (vagal nerve stimulation). METHODS After approval from the institutional review board, eight sheep were anesthetized with pentobarbital, paralyzed, and ventilated. After left thoracotomy, the bronchial artery was cannulated and perfused. In random order, 5 mg/ml concentrations of propofol, ketamine, and thiopental were infused into the bronchial artery at rates of 0.06, 0.20, and 0.60 ml/min. After 10 min, airway resistance was measured before and after vagal nerve stimulation and methacholine given via the bronchial artery. Data were expressed as a percent of baseline response before infusion of drug and analyzed by analysis of variance with significance set at P< or =0.05. RESULTS Systemic blood pressure was not affected by any of the drugs (P>0.46). Baseline airway resistance was not different among the three agents (P = 0.56) or by dose (P = 0.96). Infusion of propofol and ketamine into the bronchial artery caused a dose-dependent attenuation of the vagal nerve stimulation-induced bronchoconstriction to 26+/-11% and 8+/-2% of maximum, respectively (P<0.0001). In addition, propofol caused a significant decrease in the methacholine-induced bronchoconstriction to 43+/-27% of maximum at the highest concentration (P = 0.05) CONCLUSIONS The local bronchoprotective effects of ketamine and propofol on airways is through neurally mediated mechanisms. Although the direct effects on airway smooth muscle occur at high concentrations, these are unlikely to be of primary clinical relevance.

Efficacy of Propofol to prevent bronchoconstriction : Effects of preservative

Background The authors previously showed that propofol attenuates bronchoconstriction. Recently, a newer formulation of propofol with metabisulfite preservative has been introduced. metabisulfite causes airway narrowing in asthmatics. Therefore, we tested whether the preservative metabisulfite abolishes the ability of propofol to attenuate bronchoconstriction. The authors used a sheep model in which anesthetic agents could be directly administered to the airways via the bronchial artery. Methods After Internal Review Board approval, seven sheep were anesthetized (pentobarbital 20 mg · kg−1 · h−1) and paralyzed (pancuronium 2 mg), and the lungs were ventilated. After left thoracotomy, the bronchial artery was cannulated and perfused. In random order, propofol with and without metabisulfite, lidocaine (5 mg/ml), or metabisulfite alone (0.125 mg/ml) was infused into the bronchial artery at a rate of 0.06, 0.2, or 0.6 ml/min. After 10 min, airway resistance (Raw) was measured before and after vagal nerve stimulation (30 Hz, 30-ms duration at 30 V for 9 s.) and methacholine challenge (2 &mgr;g/ml at 2 ml/min in the bronchial artery). Data were expressed as a percent of maximal response and analyzed by analysis of variance with correction and with significance accepted at P ≤ 0.05. Results Raw at baseline was not significantly different among the four drugs (P = 0.87). Infusion of lidocaine and propofol without metabisulfite into the bronchial artery caused a dose-dependent attenuation of the vagal nerve stimulation–induced bronchoconstriction (P = 0.001). Propofol with metabisulfite had no effect on vagal nerve stimulation–induced bronchoconstriction (P = 0.40). There was a significant difference in the ability of propofol without metabisulfite compared with propofol with metabisulfite to attenuate vagal nerve stimulation–induced (P = 0.0001) and methacholine-induced bronchoconstriction (P = 0.0001). Conclusion Propofol without metabisulfite and lidocaine attenuated vagal nerve stimulation–induced bronchoconstriction in a dose-dependent fashion. Propofol without metabisulfite also decreased direct airway smooth muscle constriction. The preservative used for propofol can have a dramatic effect on its ability to attenuate bronchoconstriction.

Cerebrovascular transmural pressure and autoregulation

The cerebral blood flow (CBF) response to changes in perfusion pressure mediated through decreases in arterial pressure, increases in cerebrospinal fluid (CSF) pressure and increases in jugular venous pressure was studied in anesthetized dogs. A preparation was developed in which each of the three relevant pressures could be controlled and manipulated independently of each other. In this preparation, the superior vena cava and femoral vein were cannulated and drained into a reservoir. Blood was pumped from the reservoir into the right atrium. With this system, mean arterial pressure and jugular venous pressure could be independently controlled. CSF pressure (measured in the lateral ventricle) could be manipulated via a cisternal puncture. Total and regional CBF responses to alterations in perfusion pressure were studied with the radiolabelled microsphere technique. Each hemisphere was sectioned into 13 regions: spinal cord, cerebellum, medulla, pons, midbrain, diencephalon, caudate, hippocampus, parahippocampal gyrus, and occipital, temporal, parietal and frontal lobes. Despite 30 mm Hg reductions in arterial pressure or increases in jugular venous pressure or CSF pressure, little change in CBF was observed provided the perfusion pressure (arterial pressure minus jugular venous pressure or CSF pressure depending on which pressure was of greater magnitude) was greater than the lower limit for cerebral autoregulation (approximately 60 mm Hg). However, when the perfusion pressure was reduced by any of the three different methods to levels less than 60 mm Hg (average of 48 mm Hg), a comparable reduction (25–35%) in both total and regional CBF was obtained. Thus comparable changes in the perfusion pressure gradient established by decreasing arterial pressure, increasing jugular venous pressure and increasing CSF pressure resulted in similar total and regional blood flow responses. Independent alterations of arterial and CSF pressures, and jugular venous pressure produce opposite changes in vascular transmural pressure yet result in similar CBF responses. These results show that cerebral autoregulation is a function of the perfusion pressure gradient and cannot be accounted for predominantly by myogenic mechanisms.

Hydrostatic determinants of cerebral perfusion

We examined the cerebral blood flow response to alterations in perfusion pressure mediated through decreases in mean arterial pressure, increases in cerebrospinal fluid (CSF) pressure, and increases in jugular venous (JV) pressure in 42 pentobarbital anesthetized dogs. Each of these three pressures was independently controlled. Cerebral perfusion pressure was defined as mean arterial pressure minus JV or CSF pressure, depending on which was greater. Mean hemispheric blood flow was measured with the radiolabeled microsphere technique. Despite 30-mm Hg reductions in mean arterial pressure or increases in CSF or JV pressure, CBF did not change as long as the perfusion pressure remained greater than approximately 60 mm Hg. However, whenever perfusion pressure was reduced to an average of 48 mm Hg, cerebral blood flow decreased 27% to 33%. These results demonstrate the capacity of the cerebral vascular bed to respond similarly to changes in the perfusion pressure gradient obtained by decreasing mean arterial pressure, increasing JV pressure or increasing CSF pressure, and thereby support the above definition of cerebral perfusion pressure.

Sympathetic nerve-dependent regulation of mucosal vascular tone modifies airway smooth muscle reactivity

The airways contain a dense subepithelial microvascular plexus that is involved in the supply and clearance of substances to and from the airway wall. We set out to test the hypothesis that airway smooth muscle reactivity to bronchoconstricting agents may be dependent on airway mucosal blood flow. Immunohistochemical staining identified vasoconstrictor and vasodilator nerve fibers associated with subepithelial blood vessels in the guinea pig airways. Intravital microscopy of the tracheal mucosal microvasculature in anesthetized guinea pigs revealed that blockade of α-adrenergic receptors increased baseline arteriole diameter by ~40%, whereas the α-adrenergic receptor agonist phenylephrine produced a modest (5%) vasoconstriction in excess of the baseline tone. In subsequent in vivo experiments, tracheal contractions evoked by topically applied histamine were significantly reduced (P < 0.05) and enhanced by α-adrenergic receptor blockade and activation, respectively. α-Adrenergic ligands produced similar significant (P < 0.05) effects on airway smooth muscle contractions evoked by topically administered capsaicin, intravenously administered neurokinin A, inhaled histamine, and topically administered antigen in sensitized animals. These responses were independent of any direct effect of α-adrenergic ligands on the airway smooth muscle tone. The data suggest that changes in blood flow in the vessels supplying the airways regulate the reactivity of the underlying airway smooth muscle to locally released and exogenously administered agents by regulating their clearance. We speculate that changes in mucosal vascular function or changes in neuronal regulation of the airway vasculature may contribute to airways responsiveness in disease.

Increased hyaluronan fragmentation during pulmonary ischemia

Hyaluronan (HA), a glycosaminoglycan critical to the lung extracellular matrix, has been shown to dissociate into low-molecular-weight (LMW) HA fragments following exposure to injurious stimuli. In the present study we questioned whether lung HA changed during ischemia and whether changes had an effect on subsequent angiogenesis. After left pulmonary artery ligation (LPAL) in mice, we analyzed left lung homogenates immediately after the onset of ischemia (0 h) and intermittently for 14 days. The relative expression of HA synthase (HAS)1, HAS2, and HAS3 was determined by real-time RT-PCR, total HA in the lung was measured by an ELISA-like assay, gel electrophoresis was performed to determine changes in HA size distribution, and the activity of hyaluronidases was determined by zymography. A 50% increase in total HA was measured 16 h after the onset of ischemia and remained elevated for up to 7 days. Furthermore, a fourfold increase in LMW HA fragments (495-30 kDa) was observed by 4 h after LPAL. Both HAS1 and HAS2 showed increased expression 4-16 h after LPAL, yet no changes were seen in hyaluronidase activity. These results suggest that both HA fragmentation and activation of HA synthesis contribute to increased HA levels during lung ischemia. Delivery of LMW HA fragments in an in vitro tube formation assay or directly to the ischemic mouse lung in vivo both resulted in increased angiogenesis. We conclude that ischemic injury results in matrix fragmentation, which leads to stimulation of neovascularization.

Site of functional bronchopulmonary anastomoses in sheep.

The location of bronchopulmonary anastomoses has long been a topic of discussion, and pre‐, post‐, and capillary sites have all been demonstrated in postmortem examinations. However, there have been few studies that have provided insight into the patency and function of these anastomoses in the intact lung. To identify these functional sites where the bronchial circulation anastomoses with the pulmonary circulation, we studied sheep lungs in situ serial sectioned with high‐resolution computed tomography (CT). Differences in radiodensities of blood, air, and nonionic contrast medium were used to differentiate and localize airways and vessels and to identify the effluent from the bronchial circulation. After an initial series of scans to identify the pulmonary arteries and veins adjacent to airways 2–12 mm in diameter, contrast material was infused into the bronchial artery. In three sheep, the major accumulation of contrast medium was found in pulmonary veins. In one of the sheep, a comparable number of pulmonary arteries and veins contained contrast medium. Serial histologic sections were able to identify small bronchial venules lying within subepithelial bronchial folds that drain directly into pulmonary veins. These results using serial CT and histologic images suggest that drainage from the intraparenchymal bronchial vasculature is predominantly into postcapillary pulmonary vessels. Anat Rec 254:360–366, 1999.

Inhibition of CXCR2 attenuates bronchial angiogenesis in the ischemic rat lung

Under conditions of chronic pulmonary ischemia, the bronchial circulation undergoes massive proliferation. However, little is known regarding the mechanisms that promote neovascularization. An expanding body of literature implicates the glutamic acid-leucine-arginine (ELR+) CXC chemokines and their G protein-coupled receptor, CXCR(2), as key proangiogenic components in the lung. We used a rat model of chronic pulmonary ischemia induced by left pulmonary artery ligation (LPAL) to study bronchial angiogenesis. Using a methacrylate mixture, we cast the systemic vasculature of the rat lung at weekly intervals after LPAL. Twenty-one days after LPAL, numerous large, tortuous bronchial arteries were observed surrounding the left main bronchus that penetrated the left lung parenchyma. In stark contrast, the right lung was essentially devoid of vessels. We quantified bronchial neovascularization using 15-microm radiolabeled microspheres to measure systemic blood flow to the left lung (n = 12 rats). Results showed that by 21 days after LPAL, bronchial blood flow to the ischemic left lung had increased >10-fold compared with controls 2 days after LPAL (P < 0.01). Focusing on the predominant rat CXC chemokine that signals through CXCR(2), we measured increased levels of cytokine-induced neutrophil chemoattractant-3 protein expression in left lung homogenates early (4 and 24 h; n = 10 rats) after LPAL relative to paired right lung controls (P < 0.01). Treatment with a neutralizing antibody to CXCR(2) resulted in a significant decrease in neovascularization 21 days after LPAL (n = 9 rats; P < 0.01). Our results confirm the time course of bronchial angiogenesis in the rat and suggest the importance of CXC chemokines in promoting systemic neovascularization in the lung.

Effects of Cerebral Venous and Cerebrospinal Fluid Pressure on Cerebral Blood Flow

Regional cerebral blood flow responses to elevated jugular venous or cerebrospinal fluid pressure (Pcsf) were measured in thirty-nine anaesthetized ventilated dogs. When jugular venous pressure was elevated, arterial blood pressure and Pcsf were maintained constant at control values. When Pcsf was elevated, arterial and jugular venous pressure were maintained constant at control values. Cerebral perfusion pressure was calculated as the difference between arterial pressure and Pcsf, or jugular venous pressure, whichever downstream pressure was the highest. With an elevation in Pcsf or jugular venous pressure, the cerebral circulation autoregulated and no alterations in cerebral blood flow occurred as long as perfusion pressure remained above 60 mm Hg. When cerebral perfusion pressure decreased to values below 60 mm Hg, cerebral blood flow decreased significantly. We conclude that the brain autoregulates its blood flow to changes in perfusion pressure. In addition, our results suggest that the dominant mechanism of cerebral autoregulation is metabolic, not myogenic.