Vlahakis, Nicholas E.

Focal adhesion kinase (FAK) is a key signaling protein for integrin mediated cell migration. Auto phosphorylation of FAK at tyrosine‐397 is induced upon integrin activation, which then serves as a high affinity binding site for src tyrosine kinase forming a FAK‐src complex. Fak‐Src complex is a potent regulator of subsequent downstream signaling for enhanced cell migration. Using stable lines of SW480 colon carcinoma cells expressing integrin subunit α9, we explored the importance of the FAK‐Src complex in α9β1‐dependent cell migration. We found that ligand binding of α9β1 rapidly activated src tyrosine kinase; with concomitant tyrosine phosphorylation of FAK, p130Cas and Erk MAP kinase; inhibition of src tyrosine kinase or ErkMAP kinase activity decreased α9β1‐dependent cell migration. Surprisingly, activated α9β1 induced phosphorylation of iNOS resulting in NO production and associated NO‐mediated cell migration. Src tyrosine kinase appeared to signal upstream of iNOS, Erk, FAK and p130Cas as its inhibition blocked α9β1‐dependent tyrosine phosphorylation of these molecules. FAK binding to src was however not required for integrin α9β1 induced cell migration; and FAK inhibition (by siRNA) did not alter integrin α9β1 induced src activation and phosphorylation of Erk, p130Cas, iNOS. Analysis of Chinese Hamester Overian cells that expressed intact or mutant forms of integrin α9‐subunit showed that the α9‐cytoplasmic domain was critical for α9β1 induced src tyrosine kinase activation, downstream signaling events and ultimately cell migration. Taken together these results suggest that src, in a FAK independent manner, serves as a key early signaling protein for α9β1‐dependent cell migration; it is activated at the α9 ‐cytoplasmic domain and subsequently initiates multiple signaling intermediates, including NO, to induce cell migration.

Stewart, Glenn M., Troy J. Cross, Michael J. Joyner, Steven C. Chase, Timothy Curry, Josh Lehrer-Graiwer, Kobina Dufu, Nicholas E. Vlahakis, and Bruce D. Johnson. Impact of pharmacologically left shifting the oxygen–hemoglobin dissociation curve on arterial blood gases and pulmonary gas exchange during maximal exercise in hypoxia. High Alt Med Biol . 22:249–262, 2021. Introduction: Physiological and pathological conditions, which reduce the loading of oxygen onto hemoglobin (Hb), can impair exercise capacity and cause debilitating symptoms. Accordingly, this study examined the impact of pharmacologically left shifting the oxygen–hemoglobin dissociation curve (ODC) on arterial oxygen saturation (SaO 2 ) and exercise capacity. Methods: Eight healthy subjects completed a maximal incremental exercise test in hypoxia (F I O 2 : 0.125) and normoxia (F I O 2 : 0.21) before (Day 1) and after (Day 15) daily ingestion of 900 mg of voxelotor (an oxygen/Hb affinity modulator). Pulmonary gas exchange and arterial blood gases were assessed throughout exercise and at peak. Data for a 1,500 mg daily drug dose are reported in a limited cohort ( n = 3). Results: Fourteen days of drug administration left shifted the ODC (p50 measured under standard conditions, Day 1: 28.0 ± 2.1 mmHg vs. Day 15: 26.1 ± 1.8 mmHg, p < 0.05). Throughout incremental exercise in hypoxia, SaO 2 was systematically higher after drug (peak exercise SaO 2 on Day 1: 71 ± 2 vs. Day 15: 81% ± 2%, p < 0.001), whereas oxygen extraction (Ca-vO 2 diff) and consumption (VO 2 ) were similar (peak exercise Ca-vO 2 diff on Day 1: 11.5 ± 1.7 vs. Day 15: 11.0 ± 1.8 ml/100 ml blood, p = 0.417; peak VO 2 on Day 1: 2.59 ± 0.39 vs. Day 15: 2.47 ± 0.43 l/min, p = 0.127). Throughout incremental exercise in normoxia, SaO 2 was systematically higher after drug, whereas peak VO 2 was reduced (peak exercise SaO 2 on Day 1: 93.9 ± 1.8 vs. Day 15: 95.8% ± 1.0%, p = 0.008; peak VO 2 on Day 1: 3.62 ± 0.55 vs. Day 15: 3.26 ± 52 l/min, p = 0.001). Conclusion: Pharmacologically increasing the affinity of Hb for oxygen improved SaO 2 during hypoxia without impacting exercise capacity; however, left shifting the ODC in healthy individuals appears detrimental to exercise capacity in normoxia. Left shifting the ODC to different magnitudes and under more chronic forms of hypoxia warrants further study.

Beta‐1 integrins are important mediators of cell migration but may do so through different signaling mechanisms. However, c‐src‐activation seems to be a key signaling intermediate. Nitric oxide (NO) synthesis is another novel molecule that modulates cell migration, which may be utilized during integrin mediated cell migration. We used α9 expressing SW480 colon carcinoma cells, to explore a role for src and NO in α9β1 mediated‐cell migration. We found that α9β1‐ligation leads to c‐Src phosphorylation (Tyr‐416) and Erk MAP kinase phosphorylation; phospho‐c‐src was maximum at 5 min, and phospho‐Erk maxima was at 20min. Pre‐treated cells with src and Erk kinase inhibitors, PP1 and PD98059, inhibited α9‐dependent cell motility. c‐src appeared to signal upstream of Erk since inhibition of c‐Src‐kinase blocked Erk phosphorylation. Furthermore, α9β1‐ligation enhanced NO synthesis through activation of inducible Nitric Oxide Synthase (iNOS), which was constitutively expressed in SW480 cells. Using the NO specific inhibitor L‐NAME, α9β1‐dependent cell migration was blocked. Also, enhanced iNOS phosphorylation and NO production were src kinase (but not Erk) dependent. These results suggest a novel signaling pathway for α9β1‐induced cell migration through c‐src kinase activation of iNOS and subsequent NO production.

In humans, a novel allosteric hemoglobin-oxygen affinity modulator was administered to comprehensively examine the cardiopulmonary consequences of stabilizing a portion of the available hemoglobin in a high-oxygen affinity state during submaximal exercise in normoxia and hypoxia. Oral administration of voxelotor enhanced arterial oxygen saturation during submaximal exercise without altering oxygen consumption and central hemodynamics; however, the partial pressure of arterial carbon dioxide was reduced and the partial pressure of arterial oxygen was increased implying that hyperventilation also contributed to the increase in oxygen saturation. The preservation of arterial oxygen saturation and content was particularly evident during hypoxic submaximal exercise, when arterial desaturation typically occurs, but this did not influence arterial-venous oxygen difference.

Mechanical ventilation can injure the lung, causing edema and alveolar inflammation. Interleukin-8 (IL-8) plays an important role in this inflammatory response. We postulated that cyclic cell stretch upregulates the production and release of IL-8 by human alveolar epithelium in the absence of structural cell damage or paracrine stimulation. To test this hypothesis, alveolar epithelial cells (A549 cells) were cultured on a deformable silicoelastic membrane. When stretched by 30% for up to 48 h, the cells released 49 ± 34% more IL-8 ( P < 0.001) than static controls. Smaller deformations (20% stretch) produced no consistent increase in IL-8. Stretch of 4 h duration increased IL-8 gene transcription fourfold above baseline. Stretch had no effect on cell proliferation, cell viability as assessed by51Cr release assay, or the release of granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-α. We conclude that deformation per se can trigger inflammatory signaling and that alveolar epithelial cells may be active participants in the alveolitis associated with ventilator-induced lung injury.

Mechanical ventilation with a high tidal volume results in lung injury that is characterized by blebbing and breaks both between and through alveolar epithelial cells. We developed an in vitro model to simulate ventilator-induced deformation of the alveolar basement membrane and to investigate, in a direct manner, epithelial cell responses to deforming forces. Taking advantage of the novel fluorescent properties of BODIPY lipids and the fluorescent dye FM1-43, we have shown that mechanical deformation of alveolar epithelial cells results in lipid transport to the plasma membrane. Deformation-induced lipid trafficking (DILT) was a vesicular process, rapid in onset, and was associated with a large increase in cell surface area. DILT could be demonstrated in all cells; however, only a small percentage of cells developed plasma membrane breaks that were reversible and nonlethal. Therefore, DILT was not only involved in site-directed wound repair but might also have served as a cytoprotective mechanism against plasma membrane stress failure. This study suggests that DILT is a regulatory mechanism for membrane trafficking in alveolar epithelia and provides a novel biological framework within which to consider alveolar deformation injury and repair.

In this review, we examine the hypothesis that plasma membrane stress failure is a central event in the pathophysiology of injury from alveolar overdistension. This hypothesis leads us to consider alveolar micromechanics and specifically the mechanical interactions between lung matrix and alveolar epithelial cell cytoskeleton and plasma membrane. We then explore events that are central to the regulation of plasma membrane tension and detail the lipid-trafficking responses of in vitro deformed and/or injured cells. We conclude with a reference to upregulation of stress-responsive genes after membrane injury and resealing.

Motivated by our interest in lung deformation injury, we report on the validation of a new live cell strain system. We showed that the system maintains a cell culture environment equivalent to that provided by conventional incubators and that its strain ouput was uniform and reproducible. With this system, we defined cell deformation dose (i.e., membrane strain amplitude)-cell injury response relationships in alveolar epithelial cultures and studied the effects of temperature on them. Deformation injury occurred in the form of reversible, nonlethal plasma membrane stress failure events and was quantified as the fraction of cells with uptake and retention of fluorescein-labeled dextran (FITC-Dx). The undeformed control population showed virtually no FITC-Dx uptake at any temperature, which was also true for cells strained by 3%. However, when the membrane strain was increased to 18%, ∼5% of cells experienced deformation injury at a temperature of 37°C. Moreover, at that strain, a reduction in temperature to 4°C resulted in a threefold increase in the number of cells with plasma membrane breaks (from 4.8 to 15.9%; P < 0.05). Cooling of cells to 4°C also lowered the strain threshold at which deformation injury was first seen. That is, at a 9% substratum strain, cooling to 4°C resulted in a 10-fold increase in the number of cells with FITC-Dx staining (0.7 vs. 7.5%, P < 0.05). At that temperature, A549 cells offered a 50% higher resistance to shape change (magnetic twisting cytometry measurements) than at 37°C. We conclude that the strain-injury threshold of A549 cells is reduced at low temperatures, and we consider temperature effects on plasma-membrane fluidity, cytoskeletal stiffness, and lipid trafficking as responsible mechanisms.

Estimates of calf blood flow with venous occlusion plethysmography vary widely between studies, perhaps due to the use of different plethysmographs. Consequently, we compared calf blood flow estimates at rest and during reactive hyperemia in eight healthy subjects (four men and four women) with two commonly used plethysmographs: the mercury-in-silastic (Whitney) strain gauge and Dohn air-filled cuff. To minimize technical variability, flow estimates were compared with a Whitney gauge and a Dohn cuff on opposite calves before and after 10 min of bilateral femoral arterial occlusion. To account for any differences between limbs, a second trial was conducted in which the plethysmographs were switched. Resting flows did not differ between the plethysmographs (P = 0.096), but a trend toward lower values with the Whitney was apparent. Peak flows averaged 37% lower with the Whitney (27.8 +/- 2.8 ml.dl-1.min-1) than with the Dohn plethysmograph (44.4 +/- 2.8 ml.dl-1.min-1; P < 0.05). Peak flow expressed as a multiple above baseline was also lower with the Whitney (10-fold) than with the Dohn plethysmograph (14.5-fold; P = 0.02). Across all flows at rest and during reactive hyperemia, estimates were highly correlated between the plethysmographs in all subjects (r2 = 0.96-0.99). However, the mean slope for the Whitney-Dohn relationship was only 60 +/- 2%, indicating that over a wide range of flows the Whitney gauge estimate was 40% lower than that for the Dohn cuff. These results demonstrate that the same qualitative results can be obtained with either plethysmograph but that absolute flow values will generally be lower with Whitney gauges.