Heat stress increases carbohydrate oxidation rates and oxygen uptake during prolonged load carriage exercise.

Military missions are conducted in a multitude of environments including heat and may involve walking under load following severe exertion, the metabolic demands of which may have nutritional implications for fueling and recovery planning. Ten males equipped a military pack loaded to 30% of their body mass and walked in 20°C/40% relative humidity (RH) (TEMP) or 37°C/20% RH (HOT) either continuously (CW) for 90 min at the first ventilatory threshold or mixed walking (MW) with unloaded running intervals above the second ventilatory threshold between min 35 and 55 of the 90 min bout. Pulmonary gas, thermoregulatory, and cardiovascular variables were analyzed following running intervals. Final rectal temperature (MW: p < 0.001, g = 3.81, CW: p < 0.001, g = 4.04), oxygen uptake, cardiovascular strain, and energy expenditure were higher during HOT trials (p ≤ 0.05) regardless of exercise type. Both HOT trials elicited higher final carbohydrate oxidation (CHOox) than TEMP CW at min 90 (HOT MW: p < 0.001, g = 1.45, HOT CW: p = 0.009, g = 0.67) and HOT MW CHOox exceeded TEMP MW at min 80 and 90 (p = 0.049, g = 0.60 and p = 0.024, g = 0.73, respectively). There were no within-environment differences in substrate oxidation indicating that severe exertion work cycles did not produce a carryover effect during subsequent loaded walking. The rate of CHOox during 90 minutes of load carriage in the heat appears to be primarily affected by accumulated thermal load.

Resistance Training in the Heat: Mechanisms of Hypertrophy and Performance Enhancement.

ABSTRACT: Pryor, JL, Sweet, D, Rosbrook, P, Qiao, J, Hess, HW, and Looney, DP. Resistance training in the heat: Mechanisms of hypertrophy and performance enhancement. J Strength Cond Res 38(7): 1350-1357, 2024-The addition of heat stress to resistance exercise or heated resistance exercise (HRE) is growing in popularity as emerging evidence indicates altered neuromuscular function and an amplification of several mechanistic targets of protein synthesis. Studies demonstrating increased protein synthesis activity have shown temperature-dependent mammalian target of rapamycin phosphorylation, supplemental calcium release, augmented heat shock protein expression, and altered immune and hormone activity. These intriguing observations have largely stemmed from myotube, isolated muscle fiber, or rodent models using passive heating alone or in combination with immobilization or injury models. A growing number of translational studies in humans show comparable results employing local tissue or whole-body heat with and without resistance exercise. While few, these translational studies are immensely valuable as they are most applicable to sport and exercise. As such, this brief narrative review aims to discuss evidence primarily from human HRE studies detailing the neuromuscular, hormonal, and molecular responses to HRE and subsequent strength and hypertrophy adaptations. Much remains unknown in this exciting new area of inquiry from both a mechanistic and functional perspective warranting continued research.

Load-Velocity Profiles Before and After Heated Resistance Exercise.

ABSTRACT: Sweet, DK, Qiao, J, Rosbrook, P, and Pryor, JL. Load-velocity profiles before and after heated resistance exercise. J Strength Cond Res 38(6): 1019-1024, 2024-This study examined neuromuscular performance using load-velocity (L-V) profiles in men and women before and after resistance exercise (RE) in hot (HOT; 40° C) and temperate (TEMP; 21° C) environments. Sixteen (f = 8, m = 8) resistance-trained individuals completed a single 70-minute whole-body high-volume load (6 exercises, 4 sets of 10 repetitions) RE bout in HOT and TEMP. Before and after RE, rectal temperature (TRE), muscle temperature of the vastus lateralis (TVL) and triceps brachii (TTB), and an L-V profile for the deadlift and bench press were recorded. Thermoregulatory and L-V data were analyzed using separate 2-way repeated measures analysis of variances (ANOVAs; condition [hot, temperate] and time [pre, post]) with significance level set at p ≤ 0.05. Deadlift peak velocity was reduced at 60% 1 repetition maximum (1RM) after RE in HOT but not TEMP. Peak velocity of 40% 1RM bench press was lower in TEMP vs. HOT pre-RE (p < 0.01). Peak velocity was decreased at all loads in the deadlift L-V profile after RE, regardless of condition. Despite elevated TRE (TEMP; 37.58 ± 0.35, HOT; 38.20 ± 0.39° C), TVL (TEMP; 35.24 ± 0.62, HOT; 37.92 ± 0.55° C), and TTB (TEMP; 35.05 ± 0.78, HOT; 38.00 ± 0.16° C) after RE in HOT vs. TEMP (p < 0.01), RE in HOT did not broadly affect L-V profiles. This indicates heated resistance exercise can be performed with high-volume load and high ambient temperature with minimal performance impairment.

Endocrine Responses to Heated Resistance Exercise in Men and Women.

ABSTRACT: Pryor, JL, Sweet, DK, Rosbrook, P, Qiao, J, Looney, DP, Mahmood, S, and Rideout, T. Endocrine responses to heated resistance exercise in men and women. J Strength Cond Res 38(7): 1248-1255, 2024-We examined the endocrine responses of 16 (female = 8) resistance trained volunteers to a single bout of whole-body high-volume load resistance exercise in hot (HOT; 40° C) and temperate (TEMP; 20° C) environmental conditions. Thermoregulatory and heart rate (HR) data were recorded, and venous blood was acquired before and after resistance exercise to assess serum anabolic and catabolic hormones. In men, testosterone increased after resistance exercise in HOT and TEMP ( p < 0.01), but postexercise testosterone was not different between condition ( p = 0.51). In women, human growth hormone was different between condition at pre-exercise ( p = 0.02) and postexercise ( p = 0.03). After controlling for pre-exercise values, the between-condition postexercise difference was abolished ( p = 0.16). There were no differences in insulin-like growth factor-1 for either sex ( p ≥ 0.06). In women, cortisol increased from pre-exercise to postexercise in HOT ( p = 0.04) but not TEMP ( p = 0.19), generating a between-condition difference at postexercise ( p < 0.01). In men, cortisol increased from pre-exercise to postexercise in HOT only ( p < 0.01). Rectal temperature increased to a greater extent in HOT compared with TEMP in both men ( p = 0.01) and women ( p = 0.02). Heart rate increased after exercise under both conditions in men and women ( p = 0.01), but only women experience greater postexercise HR in HOT vs. TEMP ( p = 0.04). The addition of heat stress to resistance exercise session did not overtly shift the endocrine response toward an anabolic or catabolic response. When acute program variables are prescribed to increase postresistance exercise anabolic hormones, adding heat stress is not synergistic but does increase physiologic strain (i.e., elevated HR and rectal temperature).

Thermoregulation during a six-hour exposure to warm, humid hyperbaric conditions.

Purpose: In a disabled submarine scenario, a pressurized rescue module (PRM) may be deployed to rescue survivors. If the PRM were to become disabled, conditions could become hot and humid exposing the occupants to heat stress. We tested the hypothesis that the rise in core temperature and fluid loss from sweating would increase with rising dry bulb temperature. Methods: Twelve males (age 22 ± 3 years; height 179 ± 7 cm; mass 77.4 ± 8.3 kg) completed this study. On three occasions, subjects were exposed to high humidity and either 28-, 32-, or 35˚C for six hours in a dry hyperbaric chamber pressurized to 6.1 msw. Changes in core temperature (Tc) and body mass were recorded and linear regression lines fit to estimate the predicted rise in Tc and loss of fluid from sweating. Results: Heart rate was higher in the 35°C condition compared to the 28°C and 32°C conditions. Tc was higher in the 32°C condition compared to 28°C and higher in 35°C compared to the 28˚°C and 32°C conditions. Projected fluid loss in all of the tested conditions could exceed 6% of body mass after 24 hours of exposure endangering the health of sailors in a DISSUB or disabled PRM. A fluid intake of 1.0 to 3.5 L would be required to limit dehydration to 2% or 4% of initial mass depending upon condition. Conclusions: Prolonged exposure to 35°C conditions under pressure results in uncompensable heat stress. 32°C and 35°C exposures were compensable under these conditions but further research is required to elucidate the effect of increased ambient pressure on thermoregulation.

Lymphoedema conditions disrupt endothelial barrier function in vitro.

Lymphatic vessel contractions generate net antegrade pulsatile lymph flow. By contrast, impaired lymphatic vessels are often associated with lymphoedema and altered lymph flow. The effect of lymphoedema on the lymph flow field and endothelium is not completely known. Here, we characterized the lymphatic flow field of a platelet-specific receptor C-type lectin-like receptor 2 (CLEC2) deficient lymphoedema mouse model. In regions of lymphoedema, collecting vessels were significantly distended, vessel contractility was greatly diminished and pulsatile lymph flow was replaced by quasi-steady flow. In vitro exposure of human dermal lymphatic endothelial cells (LECs) to lymphoedema-like quasi-steady flow conditions increased intercellular gap formation and permeability in comparison to normal pulsatile lymph flow. In the absence of flow, LECs exposed to steady pressure (SP) increased intercellular gap formation in contrast with pulsatile pressure (PP). The absence of pulsatility in steady fluid flow and SP conditions without flow-induced upregulation of myosin light chain (MLCs) regulatory subunits 9 and 12B mRNA expression and phosphorylation of MLCs, in contrast with pulsatile flow and PP without flow. These studies reveal that the loss of pulsatility, which can occur with lymphoedema, causes LEC contraction and an increase in intercellular gap formation mediated by MLC phosphorylation.

Mechanical forces regulate endothelial-to-mesenchymal transition and atherosclerosis via an Alk5-Shc mechanotransduction pathway.

The response of endothelial cells to mechanical forces is a critical determinant of vascular health. Vascular pathologies, such as atherosclerosis, characterized by abnormal mechanical forces are frequently accompanied by endothelial-to-mesenchymal transition (EndMT). However, how forces affect the mechanotransduction pathways controlling cellular plasticity, inflammation, and, ultimately, vessel pathology is poorly understood. Here, we identify a mechanoreceptor that is sui generis for EndMT and unveil a molecular Alk5-Shc pathway that leads to EndMT and atherosclerosis. Depletion of Alk5 abrogates shear stress-induced EndMT responses, and genetic targeting of endothelial Shc reduces EndMT and atherosclerosis in areas of disturbed flow. Tensional force and reconstitution experiments reveal a mechanosensory function for Alk5 in EndMT signaling that is unique and independent of other mechanosensors. Our findings are of fundamental importance for understanding how mechanical forces regulate biochemical signaling, cell plasticity, and vascular disease.

Lymphatic Valves Separate Lymph Flow Into a Central Stream and a Slow-Moving Peri-Valvular Milieu.

The lymphatic system plays a pivotal role in the transport of fats, waste, and immune cells, while also serving as a metastatic route for select cancers. Using live imaging and particle tracking, we experimentally characterized the lymph flow field distal from the inguinal lymph node in the vicinity of normal bileaflet and malformed unileaflet intraluminal valves. Particle tracking experiments demonstrated that intraluminal lymphatic valves concentrate higher velocity lymph flow in the center of the vessel, while generating adjacent perivalvular recirculation zones. The recirculation zones are characterized by extended particle residence times and low wall shear stress (WSS) magnitudes in comparison to the rest of the lymphangion. A malformed unileaflet valve skewed lymph flow toward the endothelium on the vessel wall, generating a stagnation point and a much larger recirculation zone on the opposite wall. These studies define physical consequences of bileaflet and unileaflet intraluminal lymphatic valves that affect lymph transport and the generation of a heterogeneous flow field that affects the lymphatic endothelium nonuniformly. The characterized flow fields were recreated in vitro connecting different flow environments present in the lymphangion to a lymphatic endothelial cell (LEC) pro-inflammatory phenotype. Unique and detailed insight into lymphatic flow is provided, with potential applications to a variety of diseases that affect lymph transport and drug delivery.

Mechanically activated ion channel PIEZO1 is required for lymphatic valve formation.

PIEZO1 is a cation channel that is activated by mechanical forces such as fluid shear stress or membrane stretch. PIEZO1 loss-of-function mutations in patients are associated with congenital lymphedema with pleural effusion. However, the mechanistic link between PIEZO1 function and the development or function of the lymphatic system is currently unknown. Here, we analyzed two mouse lines lacking PIEZO1 in endothelial cells (via Tie2Cre or Lyve1Cre) and found that they exhibited pleural effusion and died postnatally. Strikingly, the number of lymphatic valves was dramatically reduced in these mice. Lymphatic valves are essential for ensuring proper circulation of lymph. Mechanical forces have been implicated in the development of lymphatic vasculature and valve formation, but the identity of mechanosensors involved is unknown. Expression of FOXC2 and NFATc1, transcription factors known to be required for lymphatic valve development, appeared normal in Tie2Cre;Piezo1cKO mice. However, the process of protrusion in the valve leaflets, which is associated with collective cell migration, actin polymerization, and remodeling of cell-cell junctions, was impaired in Tie2Cre;Piezo1cKO mice. Consistent with these genetic findings, activation of PIEZO1 by Yoda1 in cultured lymphatic endothelial cells induced active remodeling of actomyosin and VE-cadherin+ cell-cell adhesion sites. Our analysis provides evidence that mechanically activated ion channel PIEZO1 is a key regulator of lymphatic valve formation.

Investigating Effects of Fluid Shear Stress on Lymphatic Endothelial Cells.

Recent studies using in vivo models have characterized lymph flow and demonstrated that lymph flow plays a key role in the later stages of lymphatic vascular development, including vascular remodeling, to create a hierarchical collecting vessel network and lymphatic valves (Sweet et al., J Clin Invest 125, 2995-3007, 2015). However, mechanistic insights into the response of lymphatic endothelial cells to fluid flow are difficult to obtain from in vivo studies because of the small size of lymphatic vessels and the technical challenge of lymphatic endothelial cell isolation. On the other hand, in vitro experiments can be tailored to isolate and test specific mechanotransduction pathways more cleanly than conditions in vivo. To measure in vitro the cellular response to flow, cultured primary lymphatic endothelial cells can be exposed to highly specific fluid forces like those believed to exist in vivo. Such in vitro studies have recently helped identify FOXC2 and GATA2 as important transcriptional regulators of lymphatic function during valve formation that are regulated by lymph flow dynamics. This chapter discusses the methods used to expose primary lymphatic endothelial cells (LECs) to lymph fluid dynamics and the relationship of these in vitro studies to in vivo lymphatic biology.

Lymphovenous hemostasis and the role of platelets in regulating lymphatic flow and lymphatic vessel maturation.

Aside from the established role for platelets in regulating hemostasis and thrombosis, recent research has revealed a discrete role for platelets in the separation of the blood and lymphatic vascular systems. Platelets are activated by interaction with lymphatic endothelial cells at the lymphovenous junction, the site in the body where the lymphatic system drains into the blood vascular system, resulting in a platelet plug that, with the lymphovenous valve, prevents blood from entering the lymphatic circulation. This process, known as "lymphovenous hemostasis," is mediated by activation of platelet CLEC-2 receptors by the transmembrane ligand podoplanin expressed by lymphatic endothelial cells. Lymphovenous hemostasis is required for normal lymph flow, and mice deficient in lymphovenous hemostasis exhibit lymphedema and sometimes chylothorax phenotypes indicative of lymphatic insufficiency. Unexpectedly, the loss of lymph flow in these mice causes defects in maturation of collecting lymphatic vessels and lymphatic valve formation, uncovering an important role for fluid flow in driving endothelial cell signaling during development of collecting lymphatics. This article summarizes the current understanding of lymphovenous hemostasis and its effect on lymphatic vessel maturation and synthesizes the outstanding questions in the field, with relationship to human disease.

Lymph flow regulates collecting lymphatic vessel maturation in vivo.

Fluid shear forces have established roles in blood vascular development and function, but whether such forces similarly influence the low-flow lymphatic system is unknown. It has been difficult to test the contribution of fluid forces in vivo because mechanical or genetic perturbations that alter flow often have direct effects on vessel growth. Here, we investigated the functional role of flow in lymphatic vessel development using mice deficient for the platelet-specific receptor C-type lectin-like receptor 2 (CLEC2) as blood backfills the lymphatic network and blocks lymph flow in these animals. CLEC2-deficient animals exhibited normal growth of the primary mesenteric lymphatic plexus but failed to form valves in these vessels or remodel them into a structured, hierarchical network. Smooth muscle cell coverage (SMC coverage) of CLEC2-deficient lymphatic vessels was both premature and excessive, a phenotype identical to that observed with loss of the lymphatic endothelial transcription factor FOXC2. In vitro evaluation of lymphatic endothelial cells (LECs) revealed that low, reversing shear stress is sufficient to induce expression of genes required for lymphatic valve development and identified GATA2 as an upstream transcriptional regulator of FOXC2 and the lymphatic valve genetic program. These studies reveal that lymph flow initiates and regulates many of the key steps in collecting lymphatic vessel maturation and development.

Platelets mediate lymphovenous hemostasis to maintain blood-lymphatic separation throughout life.

Mammals transport blood through a high-pressure, closed vascular network and lymph through a low-pressure, open vascular network. These vascular networks connect at the lymphovenous (LV) junction, where lymph drains into blood and an LV valve (LVV) prevents backflow of blood into lymphatic vessels. Here we describe an essential role for platelets in preventing blood from entering the lymphatic system at the LV junction. Loss of CLEC2, a receptor that activates platelets in response to lymphatic endothelial cells, resulted in backfilling of the lymphatic network with blood from the thoracic duct (TD) in both neonatal and mature mice. Fibrin-containing platelet thrombi were observed at the LVV and in the terminal TD in wild-type mice, but not Clec2-deficient mice. Analysis of mice lacking LVVs or lymphatic valves revealed that platelet-mediated thrombus formation limits LV backflow under conditions of impaired valve function. Examination of mice lacking integrin-mediated platelet aggregation indicated that platelet aggregation stabilizes thrombi that form in the lymphatic vascular environment to prevent retrograde blood flow. Collectively, these studies unveil a newly recognized form of hemostasis that functions with the LVV to safeguard the lymphatic vascular network throughout life.

Endothelial Shc regulates arteriogenesis through dual control of arterial specification and inflammation via the notch and nuclear factor-κ-light-chain-enhancer of activated B-cell pathways.

RATIONALE: Arteriogenesis, the shear stress-driven remodeling of collateral arteries, is critical in restoring blood flow to ischemic tissue after a vascular occlusion. Our previous work has shown that the adaptor protein Shc mediates endothelial responses to shear stress in vitro. OBJECTIVE: To examine the role of the adaptor protein Shc in arteriogenesis and endothelial-dependent responses to shear stress in vivo. METHODS AND RESULTS: Conditional knockout mice in which Shc is deleted from endothelial cells were subjected to femoral artery ligation. Hindlimb perfusion recovery was attenuated in Shc conditional knockout mice compared with littermate controls. Reduced perfusion was associated with blunted collateral remodeling and reduced capillary density. Bone marrow transplantation experiments revealed that endothelial Shc is required for perfusion recovery because loss of Shc in bone marrow-derived hematopoietic cells had no effect on recovery. Mechanistically, Shc deficiency resulted in impaired activation of the nuclear factor κ-light-chain-enhancer of activated B-cell-dependent inflammatory pathway and reduced CD45⁺ cell infiltration. Unexpectedly, Shc was required for arterial specification of the remodeling arteriole by mediating upregulation of the arterial endothelial cell marker ephrinB2 and activation of the Notch pathway. In vitro experiments confirmed that Shc was required for shear stress-induced activation of the Notch pathway and downstream arterial specification through a mechanism that involves upregulation of Notch ligands delta-like 1 and delta-like 4. CONCLUSIONS: Shc mediates activation of 2 key signaling pathways that are critical for inflammation and arterial specification; collectively, these pathways contribute to arteriogenesis and the recovery of blood perfusion.

Bmper inhibits endothelial expression of inflammatory adhesion molecules and protects against atherosclerosis.

OBJECTIVE: Bone morphogenetic proteins (Bmps) are important mediators of inflammation and atherosclerosis, though their mechanism of action is not fully understood. To better understand the contribution of the Bmp signaling pathway in vascular inflammation, we investigated the role of Bmper (Bmp endothelial cell precursor-derived regulator), an extracellular Bmp modulator, in an induced in vivo model of inflammation and atherosclerosis. METHODS AND RESULTS: We crossed apolipoprotein E-deficient (ApoE(-/-)) mice with mice missing 1 allele of Bmper (Bmper(+/-) mice used in the place of Bmper(-/-) mice that die at birth) and measured the development of atherosclerosis in mice fed a high-fat diet. Bmper haploinsufficiency in ApoE(-/-) mice (Bmper(+/-);ApoE(-/-) mice) led to a more severe phenotype compared with Bmper(+/+);ApoE(-/-) mice. Bmper(+/-);ApoE(-/-) mice also exhibited increased Bmp activity in the endothelial cells in both the greater and lesser curvatures of the aortic arch, suggesting a role for Bmper in regulating Bmp-mediated inflammation associated with laminar and oscillatory shear stress. Small interfering RNA knockdown of Bmper in human umbilical vein endothelial cells caused a dramatic increase in the inflammatory markers intracellular adhesion molecule 1 and vascular cell adhesion molecule 1 at rest and after exposure to oscillatory and laminar shear stress. CONCLUSIONS: We conclude that Bmper is a critical regulator of Bmp-mediated vascular inflammation and that the fine-tuning of Bmp and Bmper levels is essential in the maintenance of normal vascular homeostasis.

The adaptor protein Shc integrates growth factor and ECM signaling during postnatal angiogenesis.

Angiogenesis requires integration of cues from growth factors, extracellular matrix (ECM) proteins, and their receptors in endothelial cells. In the present study, we show that the adaptor protein Shc is required for angiogenesis in zebrafish, mice, and cell-culture models. Shc knockdown zebrafish embryos show defects in intersegmental vessel sprouting in the trunk. Shc flox/flox; Tie2-Cre mice display reduced angiogenesis in the retinal neovascularization model and in response to VEGF in the Matrigel plug assay in vivo. Functional studies reveal a model in which Shc is required for integrin-mediated spreading and migration specifically on fibronectin, as well as endothelial cell survival in response to VEGF. Mechanistically, Shc is required for activation of the Akt pathway downstream of both integrin and VEGF signaling, as well as for integration of signals from these 2 receptors when cells are grown on fibronectin. Therefore, we have identified a unique mechanism in which signals from 2 critical angiogenic signaling axes, integrins and VEGFR-2, converge at Shc to regulate postnatal angiogenesis.

Spatial signaling networks converge at the adaptor protein Shc.

Endothelial cells, which are located at the interface between the blood and the vessel wall, respond dynamically to a variety of stimuli initiating signaling cascades that regulate cardiovascular development, physiology and pathology. These inputs include soluble factors that bind to their receptors, integrin-matrix interactions, cell-cell contacts and mechanical forces due to the flowing blood. While these stimuli can mediate unique downstream signals, it is well-accepted that signaling pathways are highly interwoven into complex signaling networks with several levels of cross-talk, integration and coordination. Recent studies suggest that several signaling networks coalesce at the adaptor protein Shc.

Shc coordinates signals from intercellular junctions and integrins to regulate flow-induced inflammation.

Atherosclerotic plaques develop in regions of the vasculature associated with chronic inflammation due to disturbed flow patterns. Endothelial phenotype modulation by flow requires the integration of numerous mechanotransduction pathways, but how this is achieved is not well understood. We show here that, in response to flow, the adaptor protein Shc is activated and associates with cell-cell and cell-matrix adhesions. Shc activation requires the tyrosine kinases vascular endothelial growth factor receptor 2 and Src. Shc activation and its vascular endothelial cadherin (VE-cadherin) association are matrix independent. In contrast, Shc binding to integrins requires VE-cadherin but occurs only on specific matrices. Silencing Shc results in reduction in both matrix-independent and matrix-dependent signals. Furthermore, Shc regulates flow-induced inflammatory signaling by activating nuclear factor kappaB-dependent signals that lead to atherogenesis. In vivo, Shc is activated in atherosclerosis-prone regions of arteries, and its activation correlates with areas of atherosclerosis. Our results support a model in which Shc orchestrates signals from cell-cell and cell-matrix adhesions to elicit flow-induced inflammatory signaling.