Building Career Paths for Ph.D., Basic and Translational Scientists in Clinical Departments in the United States: An Official American Thoracic Society Workshop Report.

Rationale: To identify barriers and opportunities for Ph.D., basic and translational scientists to be fully integrated into clinical units. Objectives: In 2022, an ad hoc committee of the American Thoracic Society developed a project proposal and workshop to identify opportunities and barriers for scientists who do not practice medicine to develop successful careers and achieve tenure-track faculty positions in clinical departments and divisions within academic medical centers (AMCs) in the United States. Methods: This document focuses on results from a survey of adult and pediatric pulmonary, critical care, and sleep medicine division chiefs as well as a survey of workshop participants, including faculty in departmental and school leadership roles in both basic science and clinical units within U.S. AMCs. Results: We conclude that full integration of non-clinically practicing basic and translational scientists into the clinical units, in addition to their traditional placements in basic science units, best serves the tripartite mission of AMCs to provide care, perform research, and educate the next generation. Evidence suggests clinical units do employ Ph.D. scientists in large numbers, but these faculty are often hired into non-tenure track positions, which do not provide the salary support, start-up funds, research independence, or space often associated with hiring in basic science units within the same institution. These barriers to success of Ph.D. faculty in clinical units are largely financial. Conclusions: Our recommendation is for AMCs to consider and explore some of our proposed strategies to accomplish the goal of integrating basic and translational scientists into clinical units in a meaningful way.

Extracellular vesicle-induced cyclic AMP signaling.

Second messenger signaling is required for cellular processes. We previously reported that extracellular vesicles (EVs) from stimulated cultured endothelial cells contain the biochemical second messenger, cAMP. In the current study, we sought to determine whether cAMP-enriched EVs induce second messenger signaling pathways in naïve recipient cells. Our results indicate that cAMP-enriched EVs increase cAMP content sufficient to stimulate PKA activity. The implications of our work are that EVs represent a novel intercellular mechanism for second messenger, specifically cAMP, signaling.

The changing face of pulmonary hypertension diagnosis: a historical perspective on the influence of diagnostics and biomarkers.

Pulmonary hypertension is a complex, multifactorial disease that results in right heart failure and premature death. Since the initial reports of pulmonary hypertension in the late 1800s, the diagnosis of pulmonary hypertension has evolved with respect to its definition, screening tools, and diagnostic techniques. This historical perspective traces the earliest roots of pulmonary hypertension detection and diagnosis through to the current recommendations for classification. We highlight the diagnostic tools used in the past and present, and end with a focus on the future directions of early detection. Early detection of pulmonary hypertension and pulmonary arterial hypertension and the proper determination of etiology are vital for the early therapeutic intervention that can prolong life expectancy and improve quality of life. The search for a non-invasive screening tool for the identification and classification of pulmonary hypertension is ongoing, and we discuss the role of animal models of the disease in this search.

Extracellular vesicles: another compartment for the second messenger, cyclic adenosine monophosphate.

The second messenger, cAMP, is highly compartmentalized to facilitate signaling specificity. Extracellular vesicles (EVs) are submicron, intact vesicles released from many cell types that can act as biomarkers or be involved in cell-to-cell communication. Although it is well recognized that EVs encapsulate functional proteins and RNAs/miRNAs, currently it is unclear whether cyclic nucleotides are encapsulated within EVs to provide an additional second messenger compartment. Using ultracentrifugation, EVs were isolated from the culture medium of unstimulated systemic and pulmonary endothelial cells. EVs were also isolated from pulmonary microvascular endothelial cells (PMVECs) following stimulation of transmembrane adenylyl cyclase (AC) in the presence or absence of the phosphodiesterase 4 inhibitor rolipram over time. Whereas cAMP was detected in EVs isolated from endothelial cells derived from different vascular beds, it was highest in EVs isolated from PMVECs. Treatment of PMVECs with agents that increase near-membrane cAMP led to an increase in cAMP within corresponding EVs, yet there was no increase in EV number. Elevated cell cAMP, measured by whole cell measurements, peaked 15 min after treatment, yet in EVs the peak increase in cAMP was delayed until 60 min after cell stimulation. Cyclic AMP was also increased in EVs collected from the perfusate of isolated rat lungs stimulated with isoproterenol and rolipram, thus corroborating cell culture findings. When added to unperturbed confluent PMVECs, EVs containing elevated cAMP were not barrier disruptive like cytosolic cAMP but maintained monolayer resistance. In conclusion, PMVECs release EVs containing cAMP, providing an additional compartment to cAMP signaling.

Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines for the field in 2014. We now update these "MISEV2014" guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

Circulating microparticles in severe pulmonary arterial hypertension increase intercellular adhesion molecule-1 expression selectively in pulmonary artery endothelium.

BACKGROUND: Microparticles (MPs) stimulate inflammatory adhesion molecule expression in systemic vascular diseases, however it is unknown whether circulating MPs stimulate localized ICAM-1 expression in the heterogeneically distinct pulmonary endothelium during pulmonary arterial hypertension (PAH). Pulmonary vascular lesions with infiltrating inflammatory cells in PAH form in the pulmonary arteries and arterioles, but not the microcirculation. Therefore, we sought to determine whether circulating MPs from PAH stimulate pulmonary artery endothelial cell-selective ICAM-1 expression. RESULTS: Pulmonary artery endothelial cells (PAECs) were exposed to MPs isolated from the circulation of a rat model of severe PAH. During late-stage (8-weeks) PAH, but not early-stage (3-weeks), an increase in ICAM-1 was observed. To determine whether PAH MP-induced ICAM-1 was selective for a specific segment of the pulmonary circulation, pulmonary microvascular endothelial cells (PMVECs) were exposed to late-stage PAH MPs and no increase in ICAM-1 was detected. A select population of circulating MPs, the late-stage endoglin + MPs, were used to assess their ability to stimulate ICAM-1 and it was determined that the endoglin + MPs were sufficient to promote ICAM-1 increases in the whole cell, but not surface only expression. CONCLUSIONS: Late-stage, but not early-stage, MPs in a model of severe PAH selectively induce ICAM-1 in pulmonary artery endothelium, but not pulmonary microcirculation. Further, the selected endoglin + PAH MPs, but not endoglin + MPs from control, are sufficient to promote whole cell ICAM-1 in PAECs. The implications of this work are that MPs in late-stage PAH are capable of inducing ICAM-1 expression selectively in the pulmonary artery. ICAM-1 likely plays a significant role in the observed inflammatory cell recruitment, specifically to vascular lesions in the pulmonary artery and not the pulmonary microcirculation.

Lectin-Based Characterization of Vascular Cell Microparticle Glycocalyx.

Microparticles (MPs) are released constitutively and from activated cells. MPs play significant roles in vascular homeostasis, injury, and as biomarkers. The unique glycocalyx on the membrane of cells has frequently been exploited to identify specific cell types, however the glycocalyx of the MPs has yet to be defined. Thus, we sought to determine whether MPs, released both constitutively and during injury, from vascular cells have a glycocalyx matching those of the parental cell type to provide information on MP origin. For these studies we used rat pulmonary microvascular and artery endothelium, pulmonary smooth muscle, and aortic endothelial cells. MPs were collected from healthy or cigarette smoke injured cells and analyzed with a panel of lectins for specific glycocalyx linkages. Intriguingly, we determined that the MPs released either constitutively or stimulated by CSE injury did not express the same glycocalyx of the parent cells. Further, the glycocalyx was not unique to any of the specific cell types studied. These data suggest that MPs from both normal and healthy vascular cells do not share the parental cell glycocalyx makeup.

Severe pulmonary arterial hypertensive rats are tolerant to mild exercise.

A frequently used end point of clinical outcomes in patients with pulmonary arterial hypertension (PAH) is the 6-minute walk distance. Furthermore, some data suggest that mild to moderate exercise as an intervention in stable PAH is beneficial. Some of these questions have been recapitulated in the monocrotaline and hypoxia animal models of pulmonary hypertension. However, mild exercise and walk distance as end points have not been rigorously examined in the severe progressive Sugen 5416/hypoxia/normoxia (Su/Hx/Nx) animal model of PAH at each stage of worsening disease. Our hypothesis was that animals that were preselected as runners would have increased walk times and improved right ventricle/left ventricle plus septum (RV/LV+S) ratios, echocardiography, and histology compared with nonexercised Su/Hx/Nx animals. We examined four groups of rats: Su/Hx/Nx sedentary, Su/Hx/Nx exercised, control sedentary, and control exercised. Echocardiography was performed at 5, 8, and 13 weeks to assess right ventricular inner diameter in diastole and left ventricular eccentricity index. We found no difference between exercised and sedentary Su/Hx/Nx rats, and both were worsened compared with controls. Rats were euthanized at 13 weeks, and we found that neither RV/LV+S nor the occurrence of occlusive lesions were influenced by exercise. Most interesting, however, was that despite progressive PAH development, exercised Su/Hx/Nx rats showed no decrease in time or distance for treadmill exercise. In all, our data suggest that, despite severe PAH development, Su/Hx/Nx rats retain the same treadmill exercise capacity as control animals.

On the origin of microparticles: From "platelet dust" to mediators of intercellular communication.

Microparticles are submicron vesicles shed from a variety of cells. Peter Wolf first identified microparticles in the midst of ongoing blood coagulation research in 1967 as a product of platelets. He termed them platelet dust. Although initially thought to be useless cellular trash, decades of research focused on the tiny vesicles have defined their roles as participators in coagulation, cellular signaling, vascular injury, and homeostasis. The purpose of this review is to highlight the science leading up to the discovery of microparticles, feature discoveries made by key contributors to the field of microparticle research, and discuss their positive and negative impact on the pulmonary circulation.

Serotonin passes through myoendothelial gap junctions to promote pulmonary arterial smooth muscle cell differentiation.

Myoendothelial gap junctional signaling mediates pulmonary arterial endothelial cell (PAEC)-induced activation of latent TGF-ß and differentiation of cocultured pulmonary arterial smooth muscle cells (PASMCs), but the nature of the signal passing from PAECs to PASMCs through the gap junctions is unknown. Because PAECs but not PASMCs synthesize serotonin, and serotonin can pass through gap junctions, we hypothesized that the monoamine is the intercellular signal. We aimed to determine whether PAEC-derived serotonin mediates PAEC-induced myoendothelial gap junction-dependent activation of TGF-ß signaling and differentiation of PASMCs. Rat PAECs and PASMCs were monocultured or cocultured with (touch) or without (no-touch) direct cell-cell contact. In all cases, tryptophan hydroxylase 1 (Tph1) transcripts were expressed predominantly in PAECs. Serotonin was detected by immunostaining in both PAECs and PASMCs in PAEC/PASMC touch coculture but was not found in PASMCs in either PAEC/PASMC no-touch coculture or in PASMC/PASMC touch coculture. Furthermore, inhibition of gap junctions but not of the serotonin transporter in PAEC/PASMC touch coculture prevented serotonin transfer from PAECs to PASMCs. Inhibition of serotonin synthesis pharmacologically or by small interfering RNAs to Tph1 in PAECs inhibited the PAEC-induced activation of TGF-ß signaling and differentiation of PASMCs. We concluded that serotonin synthesized by PAECs is transferred through myoendothelial gap junctions into PASMCs, where it activates TGF-ß signaling and induces a more differentiated phenotype. This finding suggests a novel role of gap junction-mediated intercellular serotonin signaling in regulation of PASMC phenotype.

Myoendothelial gap junctional signaling induces differentiation of pulmonary arterial smooth muscle cells.

Myoendothelial gap junctions are involved in regulating systemic arterial smooth muscle cell phenotype and function, but their role in the regulation of pulmonary arterial smooth muscle cell (PASMC) phenotype is unknown. We therefore investigated in cocultured pulmonary arterial endothelial cells (PAECs) and PASMCs whether myoendothelial gap junctional signaling played a role in PAEC-dependent regulation of PASMC phenotype. Rat PAECs and PASMCs were cocultured on opposite sides of a porous Transwell membrane that permitted formation of heterotypic cell-cell contacts. Immunostaining showed expression of the gap junctional protein connexin 43 (Cx43) on projections extending into the membrane from both cell types. Dye transfer exhibited functional gap junctional communication from PAECs to PASMCs. PASMCs cocultured with PAECs had a more contractile-like phenotype (spindle shape and increased expression of the contractile proteins myosin heavy chain, H1-calponin, and α-smooth muscle cell-actin) than PASMCs cocultured with PASMCs or cocultured without direct contact with PAECs. Transforming growth factor (TGF)-ß1 signaling was activated in PASMCs cocultured with PAECs, and the PASMC differentiation was inhibited by TGF-ß type I receptor blockade. Inhibition of gap junctional communication pharmacologically or by knock down of Cx43 in PAECs blocked TGF-ß signaling and PASMC differentiation. These results implicate myoendothelial gap junctions as a gateway for PAEC-derived signals required for maintaining TGF-ß-dependent PASMC differentiation. This study identifies an alternative pathway to paracrine signaling to convey regulatory signals from PAECs to PASMCs and raises the possibility that dysregulation of this direct interaction is involved in the pathogenesis of hypertensive pulmonary vascular remodeling.

Rac1 activity regulates proliferation of aggressive metastatic melanoma.

Molecular mechanisms underlying the different capacity of two in vivo selected human melanoma cell variants to form experimental metastases were studied. The doubling times of the FEMX-I and FEMX-V cell sublines in vitro were 15 and 25 h, respectively. The invasive capacity of FEMX-I cells was 8-fold higher than FEMX-V cells, and the time to form approximately 10 mm s.c. tumors in nude mice was 21 versus 35 days. FEMX-I displayed a spindle-like formation in vitro, whereas FEMX-V cells had a rounded shape. Hence, we examined known determinants of cell shape and proliferation, the small GTPases. The four studied showed equal expression in both cell types, but Rac1 activity was significantly decreased in FEMX-V cells. Rac1 stimulates NFkappaB, and we found that endogenous NFkappaB activity of FEMX-V cells was 2% of that of FEMX-I cells. Inhibition of Rac1 resulted in blocked NFkappaB activity. Specific inhibition of either Rac1 or NFkappaB significantly reduced proliferation and invasion of FEMX-I cells, the more pronounced effects observed with Rac1 inhibition. These data indicate that Rac1 activity in FEMX cells regulates cell proliferation and invasion, in part via its effect on NFkappaB, signifying Rac1 as a key molecule in melanoma progression and metastasis.

Platelet-derived growth factor BB induces nuclear export and proteasomal degradation of CREB via phosphatidylinositol 3-kinase/Akt signaling in pulmonary artery smooth muscle cells.

Cyclic AMP response element binding protein (CREB) content is diminished in smooth muscle cells (SMCs) in remodeled pulmonary arteries from animals with pulmonary hypertension and in the SMC layers of atherogenic systemic arteries and cardiomyocytes from hypertensive individuals. Loss of CREB can be induced in cultured SMCs by chronic exposure to hypoxia or platelet-derived growth factor BB (PDGF-BB). Here we investigated the signaling pathways and mechanisms by which PDGF elicits depletion of SMC CREB. Chronic PDGF treatment increased CREB ubiquitination in SMCs, while treatment of SMCs with the proteasome inhibitor lactacystin prevented decreases in CREB content. The nuclear export inhibitor leptomycin B also prevented depletion of SMC CREB alone or in combination with lactacystin. Subsequent studies showed that PDGF activated extracellular signal-regulated kinase, Jun N-terminal protein kinase, and phosphatidylinositol 3 (PI3)-kinase pathways in SMCs. Inhibition of these pathways blocked SMC proliferation in response to PDGF, but only inhibition of PI3-kinase or its effector, Akt, blocked PDGF-induced CREB loss. Finally, chimeric proteins containing enhanced cyan fluorescent protein linked to wild-type CREB or CREB molecules with mutations in several recognized phosphorylation sites were introduced into SMCs. PDGF treatment reduced the levels of each of these chimeric proteins except for one containing mutations in adjacent serine residues (serines 103 and 107), suggesting that CREB loss was dependent on CREB phosphorylation at these sites. We conclude that PDGF stimulates nuclear export and proteasomal degradation of CREB in SMCs via PI3-kinase/Akt signaling. These results indicate that in addition to direct phosphorylation, proteolysis and intracellular localization are key mechanisms regulating CREB content and activity in SMCs.

Putative role for a myosin motor in store-operated calcium entry.

Store-operated calcium (SOC) entry is the most prominent mode of calcium entry in nonexcitable cells, although important questions remain regarding its mechanism(s) of activation and the molecular identity of SOC entry channels. Recent work using Drosophila melanogaster and mammalian cells suggest that myosin may play a central role in regulation of the open state of SOC entry channels. The most direct evidence for such a role for myosin motor function is in the Drosophila rhabdomere, where a myosin homolog appears to terminate channel signaling. Studies directly examining the contribution of myosin to mammalian SOC entry are lacking. However, several indirect lines of evidence support a role for myosin motor function in the control of calcium entry. Both inhibition of myosin light-chain kinase (the kinase responsible for myosin activation) and disruption of filamentous actin (the track for actomyosin motor function) reduces SOC entry and appear to prevent activation of a calcium-selective SOC entry current. Thus, this review summarizes data-emphasizing recent evidence in mammalian systems-implicating myosin motor function in the control of SOC entry.