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Background Recent studies showed a connection between pyrodiversity and biodiversity in the western US, but few examined bird response to fire severity in eastern hardwood forests. We measured forest structure and conducted avian point counts for five years (2016–2021; Y1-Y5) in three burned and three unburned watersheds following mixed-severity wildfires to compare breeding bird communities across a fire-severity gradient (no burn (NB), low (L), low-moderate (LM), moderate (M), moderate-high (MH), and high (H)) in the southern Appalachian region. Results The percentage of wildfire-killed trees increased with fire-severity category (FSC) and over time; by Y5, 74% (48% BA) were dead in H compared to 11% (3% BA) in L. Post-wildfire shrub recovery was rapid, especially in H and MH where cover exceeded all other FSCs by Y5. Total bird abundance and species richness increased with increasing fire severity (no change in NB and L) and exceeded all other FSCs in H and MH by Y5. Most (12 of 18 tested) species were unaffected by wildfire severity. Disturbance-dependent chestnut-sided warbler (Setophaga pensylvanica), indigo bunting (Passerina cyanea), and eastern towhee (Pipilo erythrophthalmus) increased in H within 3–4 years; white-breasted nuthatch (Sitta carolinensis), hooded warbler (Setophaga citrina), and blue-headed vireo (Vireo solitarius) increased in some burned FSCs and years. Shrub-nesters were more abundant in H than all other FSCs except MH and increased over time in H, MH and LM. Cavity-nesters were more abundant in H, MH and M than L or NB, but primary cavity-nesters (woodpeckers) alone were not. Conclusions Our results highlight the interrelated roles of fire severity and time in driving breeding bird response in central hardwood forests, illustrated by sharp contrasts in bird responses between L (no effects) and higher-severity (H and MH) burns that promoted disturbance-dependent species, but also a more gradual increase in total bird abundance and species richness in LM and M corresponding with delayed tree mortality and shrub recovery. We suggest that high-severity burns are more important than pyrodiversity per se in promoting bird diversity, as disturbance-dependent species were mainly associated with high-severity burns whereas no species was limited to unburned forests or lower-severity burns (L-M).

Wildfires are landscape scale disturbances that can significantly affect hydrologic processes such as runoff generation and sediment and nutrient transport to streams. In Fall 2016, multiple large drought‐related wildfires burned forests across the southern Appalachian Mountains. Immediately after the fires, we identified and instrumented eight 28.4–344 ha watersheds (four burned and four unburned) to measure vegetation, soil, water quantity, and water quality responses over the following two years. Within burned watersheds, plots varied in burn severity with up to 100% tree mortality and soil O‐horizon loss. Watershed scale high burn severity extent ranged from 5% to 65% of total watershed area. Water quantity and quality responses among burned watersheds were closely related to the high burn severity extent. Total water yield (Q) was up to 39% greater in burned watersheds than unburned reference watersheds. Total suspended solids (TSS) concentration during storm events were up to 168 times greater in samples collected from the most severely burned watershed than from a corresponding unburned reference watershed, suggesting that there was elevated risk of localized erosion and sedimentation of streams. NO₃‐N concentration, export, and concentration dependence on streamflow were greater in burned watersheds and increased with increasing high burn severity extent. Mean NO₃‐N concentration in the most severely burned watershed increased from 0.087 mg L⁻¹ in the first year to 0.363 mg L⁻¹ (+317%) in the second year. These results suggest that the 2016 wildfires degraded forest condition, increased Q, and had negative effects on water quality particularly during storm events.

In this study, we have looked for an optimum media glucose concentration and compared glucose consumption in three vascular cell types, endothelial cells (ECs), vascular smooth muscle cells (VSMCs), and adventitial fibroblasts (AFs) with or without angiotensin II (AngII) stimulation. In a subconfluent 6-well experiment in 1 mL DMEM with a standard low (100 mg/dL), a standard high (450 mg/dL), or a mixed middle (275 mg/dL) glucose concentration, steady and significant glucose consumption was observed in all cell types. After 48-h incubation, media that contained low glucose was reduced to almost 0 mg/dL, media that contained high glucose remained significantly higher at ∼275 mg/dL, and media that contained middle glucose remained closer to physiological range. AngII treatment enhanced glucose consumption in AFs and VSMCs but not in ECs. Enhanced extracellular acidification rate by AngII was also observed in AFs. In AFs, AngII induction of target proteins at 48 h varied depending on the glucose concentration used. In low glucose media, induction of glucose regulatory protein 78 or hexokinase II was highest, whereas induction of VCAM-1 was lowest. Utilization of specific inhibitors further suggests essential roles of angiotensin II type-1 receptor and glycolysis in AngII-induced fibroblast activation. Overall, this study demonstrates a high risk of hypo- or hyperglycemic conditions when standard low or high glucose media is used with vascular cells. Moreover, these conditions may significantly alter experimental outcomes. Media glucose concentration should be monitored during any culture experiments and utilization of middle glucose media is recommended for all vascular cell types.

Rhododendron maximum, a native ericaceous evergreen shrub, is expanding in forests of the southern Appalachian region following eastern hemlock (Tsuga canadensis) mortality due to hemlock woolly adelgid (Adelges tsugae) infestations. The goal of our study was to examine soil microbial community responses to experimental R. maximum removal treatments. The experiment was implemented as a 2 x 2 factorial design, including two R. maximum canopy removal levels (cut vs. not cut) combined with two forest floor removal levels (burned vs. not burned). These treatments were designed as potential management strategies to facilitate hardwood tree establishment in forests that have experienced T. canadensis declines. We sampled soils after removals and characterized bacterial and fungal communities using amplicon sequencing. Shrub removal did not affect bacterial or fungal alpha diversity but did affect both bacterial and fungal community composition. Relative abundances of bacterial phyla and fungal classes exhibited no differences among R. maximum removal treatments. However, specific bacterial and fungal taxa that were responsive to R. maximum removal (i.e., differentially abundant sequences) did exhibit clear patterns at high taxonomic levels. Specifically, taxa that responded negatively to R. maximum removal were found primarily in two bacterial phyla (Proteobacteria and Bacteroidetes) and one fungal class (Archaeorhizomycetes) while positive responders were clustered in several other bacterial phyla (e.g., Actinobacteria, Planctomycetes, Cyanobacteria). Fungal functional guilds also responded to R. maximum removal, including negative responses of ericoid mycorrhizae and positive responses of arbuscular mycorrhizae and wood saprotrophs. Effects of R. maximum removal on soil microbial communities were minor overall, but clear effects on some key functional groups were evident (i.e., mycorrhizal fungi), suggesting that microbial responses to R. maximum removal may influence recovery of forests in the southern Appalachian region. Coweeta LTER - National Science Foundation [DEB-1637522]; Coweeta Hydrologic Laboratory, Southern Research Station, USDA Forest Service Published version This project was supported by the Coweeta LTER, funded by National Science Foundation grant DEB-1637522. We thank the Coweeta Hydrologic Laboratory, Southern Research Station, USDA Forest Service for support and the USDA Forest Service Nantahala Ranger District staff, Nantahala National Forest, for executing the treatments. We also thank Bobbie Niederlehner for help with soil chemistry analyses. The use of trade or firm names in this publication is for reader information and does not imply endorsement by the U.S. Department of Agriculture of any product or service. Public domain – authored by a U.S. government employee

Prior to the 1950s, common partial harvesting operations in the southern Appalachians USA involved the removal of logs by ground-skidding and the construction of steep access roads and skid trails along stream channels. Little is known about how these historical practices affected long-term vegetation changes. An experimental watershed in the Coweeta Hydrologic Laboratory was partially harvested in the years 1942–1952 according to these exploitive practices. We compared the partial-cut watershed to a clear-cut watershed and an untreated, reference watershed. Using long-term vegetation surveys, we analyzed patterns in aboveground biomass accumulation, species composition and diversity (Shannon’s index Hʹ and species richness) among watersheds. Contrary to our expectations, the partial-cut watershed recovered to reference levels of aboveground biomass and their species composition was similar over time. The clear-cut watershed had greater abundance of tulip poplar (Liriodendron tulipifera) and black locust (Robinia pseudoacacia) than the other two watersheds. The partial-cut watershed increased in diversity over time, but it remained less diverse than the reference watershed; whereas, the clear-cut watershed had lower diversity than the other two watersheds, and its diversity did not change over time. Distributions of functional groups based on water use and nutrient acquisition traits, and shade tolerance were similar between the partial-cut and the reference watersheds, but differed from the clear-cut watershed. By the 2010s, partial-cut and reference watersheds had similar proportions of diffuse-porous (32% and 33%) and ring-porous (48% and 42%) basal area, while the clear-cut watershed had more diffuse-porous (43%) and less ring-porous (36%) species than the partial-cut or reference watersheds. Tree species associated with arbuscular mycorrhiza were more abundant in the clear-cut watershed than the partial-cut or reference watersheds. Overall, the partial-cut watershed, even with the extreme soil disturbance, did not alter long-term species composition and diversity as dramatically as the clear-cut watershed. These results could help forest managers, conservationists, and hydrologists understand the long-term effects of partial-cutting versus clear-cutting.

The loss of Tsuga canadensis from invasion by hemlock woolly adelgid (Adelges tsugae, HWA) has altered ecosystem structure and function in forests across the eastern United States. In southern Appalachian forests, T. canadensis co-occurred with hardwood species and an evergreen Rhododendron maximum shrub layer in riparian and cove positions. In this region, HWA infestation was detected in 2003, with mortality reaching 97% by 2014. In this study we examined responses of light, soil moisture, tree seedling density, and overstory and understory vegetation growth from 2004 to 2014 following HWA infestation and mortality of T. canadensis. We hypothesized that seedling recruitment and vegetation growth would continue to increase over time as observed with initial trends reported through 2009, and that species that associate with ectomycorrhizal (ECM) fungi may benefit more than those that do not due to the pulse of organic material in these stands from dead T. canadensis trees. Light transmission measured at 1 m above the ground increased from 2006 to 2009, but gradually decreased from 2015 to 2017. Basal area of overstory non-Tsuga trees increased only marginally, and there was no recruitment of tree seedlings to the overstory size class, even though seedling density of deciduous species increased initially. Increased basal area and stem density of R. maximum may explain the light and seedling responses, as this species can inhibit tree seedling recruitment by limiting light and nutrients. Overstory species with the highest basal area increment (BAI) in the post-T. canadensis stands were Pinus rigida, Betula lenta and Quercus coccinea, which all associate with ECM fungi. However, not all ECM tree species grew significantly more following T. canadensis mortality compared to pre-mortality growth rates—only those ECM species that had high growth rates prior to mortality did. After a decade, growth of both overstory trees and R. maximum has not compensated for the loss of T. canadensis. Active management of R. maximum, which may involve the removal of the evergreen shrub and soil organic layer, may be required to allow for diverse tree seedling recruitment; and subsequently, restore riparian forest structure, diversity, and function.

Forest ecosystems dominated by Tsuga canadensis are undergoing fundamental changes in function and composition from infestations by hemlock woolly adelgid (Adelges tsugae). We proposed that the first step to restoring southern Appalachian riparian forests following T. canadensis mortality would be eliminating the evergreen shrub, Rhododendron maximum. We hypothesized that removing R. maximum would increase light transmittance, soil moisture and temperature; and subsequently, enhance herbaceous-layer diversity and promote tree seedling recruitment and survival. We tested these hypotheses at two locations, (CWT, Coweeta Hydrologic Laboratory; WOC, White Oak Creek) in the Nantahala Mountain Range of western North Carolina, both with heavy T. canadensis mortality and a dense R. maximum subcanopy. The treatments were designed to remove only soil O-horizon (FF), remove only R. maximum (CR), remove R. maximum and soil O-horizon (CFFR), and untreated, reference (REF). We installed permanent plots across treatments and locations and measured light transmittance (Qi/Qo), soil water content (θ), herbaceous-layer cover and diversity (Shannon’s index (H′cover) and species richness), and tree seedling recruitment. As expected, cutting the R. maximum subcanopy (CR and CFFR) immediately increased Qi/Qo in the spring months across locations, and it was sustained through the first growing season. θ was generally high across plots, averaging 26% during the growing season, and didn’t vary over time. By the second growing season (2017) after treatments, herbaceous-layer cover and diversity increased on CR and CFFR. Herbaceous-layer cover was significantly related to Qi/Qo (r2 = 0.22, p Acer rubrum > Quercus coccinea > Liriodendron tulipifera > Q. rubra. In CFFR, tree seedling recruitment ranked Betula spp. > A. rubrum > L. tulipifera. These vegetation responses have important implications for potential recovery of riparian forests following T. canadensis mortality.

A disintegrin and metalloproteases (ADAMs) are key mediators of cell signaling by ectodomain shedding of various growth factors, cytokines, receptors and adhesion molecules at the cellular membrane. ADAMs regulate cell proliferation, cell growth, inflammation, and other regular cellular processes. ADAM17, the most extensively studied ADAM family member, is also known as tumor necrosis factor (TNF)-α converting enzyme (TACE). ADAMs-mediated shedding of cytokines such as TNF-α orchestrates immune system or inflammatory cascades and ADAMs-mediated shedding of growth factors causes cell growth or proliferation by transactivation of the growth factor receptors including epidermal growth factor receptor. Therefore, increased ADAMs-mediated shedding can induce inflammation, tissue remodeling and dysfunction associated with various cardiovascular diseases such as hypertension and atherosclerosis, and ADAMs can be a potential therapeutic target in these diseases. In this review, we focus on the role of ADAMs in cardiovascular pathophysiology and cardiovascular diseases. The main aim of this review is to stimulate new interest in this area by highlighting remarkable evidence.

Cardiovascular disease (CVD) is a prevalent issue in the global aging population. Premature vascular aging such as elevated arterial stiffness appears to be a major risk factor for CVD. Vascular smooth muscle cells (VSMCs) are one of the essential parts of arterial pathology and prone to stress-induced senescence. The pervasiveness of senescent VSMCs in the vasculature increases with age and can be further expedited by various stressing events such as oxidative stress, mitochondria dysfunction, endoplasmic reticulum stress, and chronic inflammation. Angiotensin II (AngII) can induce many of these responses in VSMCs and is thus considered a key regulator of VSMC senescence associated with CVD. Understanding the precise mechanisms and consequences of senescent cell accumulation may uncover a new generation of therapies including senolytic and senomorphic compounds against CVD. Accordingly, in this review article, we discuss potential molecular mechanisms of VSMC senescence such as those induced by AngII and the therapeutic manipulations of senescence to control age-related CVD and associated conditions such as by senolytic.

Investigations of vascular smooth muscle cell (VSMC) phenotypic modulation due to angiotensin II (AngII) stimulation are important for understanding molecular mechanisms contributing to hypertension and associated vascular pathology. AngII induces endoplasmic reticulum (ER) stress in VSMCs, which has been implicated in hypertensive vascular remodeling. Under ER stress, 78 kDa glucose-regulated protein (GRP78) acts as an endogenous chaperone, as well as a master controller of unfolded protein response (UPR) to maintain protein quality control. However, the potential downstream consequences of ER stress induced by AngII on protein quality control and pro-inflammatory phenotype in VSMCs remain elusive. This study aims to identify protein aggregation as evidence of the disruption of protein quality control in VSMCs, and to test the hypothesis that preservation of proteostasis by overexpression of GRP78 can attenuate the AngII-induced pro-inflammatory phenotype in VSMCs. Increases in protein aggregation and enhanced UPR were observed in VSMCs exposed to AngII, which were mitigated by overexpression of GRP78. Moreover, GRP78 overexpression attenuated enhanced monocyte adhesion to VSMCs induced by AngII. Our results thus indicate that the prevention of protein aggregation can potentially mitigate an inflammatory phenotype in VSMCs, which may suggest an alternative therapy for the treatment of AngII-associated vascular disorders.

Endothelial inflammation and mitochondrial dysfunction have been implicated in cardiovascular diseases, yet, a unifying mechanism tying them together remains limited. Mitochondrial dysfunction is frequently associated with mitochondrial fission/fragmentation mediated by the GTPase Drp1 (dynamin-related protein 1). Nuclear factor (NF)-κB, a master regulator of inflammation, is implicated in endothelial dysfunction and resultant complications. Here, we explore a causal relationship between mitochondrial fission and NF-κB activation in endothelial inflammatory responses. In cultured endothelial cells, TNF-α (tumor necrosis factor-α) or lipopolysaccharide induces mitochondrial fragmentation. Inhibition of Drp1 activity or expression suppresses mitochondrial fission, NF-κB activation, vascular cell adhesion molecule-1 induction, and leukocyte adhesion induced by these proinflammatory factors. Moreover, attenuations of inflammatory leukocyte adhesion were observed in Drp1 heterodeficient mice as well as endothelial Drp1 silenced mice. Intriguingly, inhibition of the canonical NF-κB signaling suppresses endothelial mitochondrial fission. Mechanistically, NF-κB p65/RelA seems to mediate inflammatory mitochondrial fission in endothelial cells. In addition, the classical anti-inflammatory drug, salicylate, seems to maintain mitochondrial fission/fusion balance against TNF-α via inhibition of NF-κB. In conclusion, our results suggest a previously unknown mechanism whereby the canonical NF-κB cascade and a mitochondrial fission pathway interdependently regulate endothelial inflammation.

The transformation of vascular smooth muscle cells [VSMC] into foam cells leading to increased plaque size and decreased stability is a key, yet understudied step in atherogenesis. We reported that Interleukin-19 (IL-19), a novel, anti-inflammatory cytokine, attenuates atherosclerosis by anti-inflammatory effects on VSMC. In this work we report that IL-19 induces expression of miR133a, a muscle-specific miRNA, in VSMC. Although previously unreported, we report that miR133a can target and reduce mRNA abundance, mRNA stability, and protein expression of Low Density Lipoprotein Receptor Adaptor Protein 1, (LDLRAP1), an adaptor protein which functions to internalize the LDL receptor. Mutations in this gene lead to LDL receptor malfunction and cause the Autosomal Recessive Hypercholesterolemia (ARH) disorder in humans. Herein we show that IL-19 reduces lipid accumulation in VSMC, and LDLRAP1 expression and oxLDL uptake in a miR133a-dependent mechanism. We show that LDLRAP1 is expressed in plaque and neointimal VSMC of mouse and human injured arteries. Transfection of miR133a and LDLRAP1 siRNA into VSMC reduces their proliferation and uptake of oxLDL. miR133a is significantly increased in plasma from hyperlipidemic compared with normolipidemic patients. Expression of miR133a in IL-19 stimulated VSMC represents a previously unrecognized link between vascular lipid metabolism and inflammation, and may represent a therapeutic opportunity to combat vascular inflammatory diseases.

It has been proposed that membrane microdomains, caveolae, in vascular cells are critical for signal transduction and downstream functions induced by angiotensin II (AngII). We have tested our hypothesis that caveolin-1 (Cav1), a major structural protein of vascular caveolae, plays a critical role in the development of vascular remodeling by AngII via regulation of epidermal growth factor receptor and vascular endothelial adhesion molecule-1. Cav1 −/− and control Cav +/+ mice were infused with AngII for 2 weeks to induce vascular remodeling and hypertension. On AngII infusion, histological assessments demonstrated medial hypertrophy and perivascular fibrosis of aorta and coronary and renal arteries in Cav1 +/+ mice compared with sham-operated Cav1 +/+ mice. AngII-infused Cav1 +/+ mice also showed a phenotype of cardiac hypertrophy with increased heart weight to body weight ratio compared with control Cav1 +/+ mice. In contrast, Cav1 −/− mice infused with AngII showed attenuation of vascular remodeling but not cardiac hypertrophy. Similar levels of AngII-induced hypertension were found in both Cav1 +/+ and Cav1 −/− mice as assessed by telemetry. In Cav1 +/+ mice, AngII enhanced tyrosine-phosphorylated epidermal growth factor receptor staining in the aorta, which was attenuated in Cav1 −/− mice infused with AngII. Enhanced Cav1 and vascular endothelial adhesion molecule-1 expression was also observed in aorta from AngII-infused Cav1 +/+ mice but not in Cav1 −/− aorta. Experiments with vascular cells further provided a potential mechanism for our in vivo findings. These data suggest that Cav1, and presumably caveolae, in vascular smooth muscle and the endothelium plays a critical role in vascular remodeling and inflammation independent of blood pressure or cardiac hypertrophy regulation.

In the past century, the evergreen woody shrub, Rhododendron maximum, has experienced habitat expansion following foundational tree species die-off in eastern US deciduous forests. Rhododendron can potentially alter stream chemistry, temperature, trophic dynamics, and in-stream decomposition rates, given its dominance in riparian areas. Here we conducted two operational-scale (3 ha) riparian treatments that removed rhododendron through cutting alone (CR, canopy removal), or removing both the rhododendron canopy and forest floor using cutting and prescribed fire (CFFR, canopy and forest floor removal). We expected that rhododendron shrub removal, with or without soil organic horizon removal, would increase soil nutrient availability and subsequently alter stream pH, acid neutralizing capacity (ANC), inorganic nitrogen (NO3-N, NH4-N), total dissolved inorganic nitrogen, dissolved organic carbon (DOC), calcium (Ca), potassium (K), and magnesium (Mg). We hypothesized that responses would occur more quickly in the CFFR treatment. Treatments reduced shrub-, but not tree basal area. Treatments lowered soil N, but not C. Stream chemistry responses to treatments varied between CR and CFFR and were transient, generally with pH, N, and some cations declining, and aluminum (Al) and DOC showing a pulse increase. By removing rhododendron, the remaining deciduous trees likely accelerated N uptake as soil moisture availability increased. This could partially explain why we observed lower than expected stream nutrients (NO3-N, Ca, and Mg) after treatments. Initial rhododendron slash on the forest floor coupled with incomplete consumption of the O-horizon on the CFFR treatment likely elevated DOC in the upper soil horizons and mobilized Al. From a management perspective, using these treatments to restore structure and function to riparian forests in the wake of eastern hemlock mortality, with or without fire, would most likely not result in short-term diminished water quality that is common when overstory trees are harvested and may even lower stream NO3-N concentrations long term.

Southern Appalachian high elevation watersheds have deep rocky soils with high organic matter content, different vegetation communities, and receive greater inputs of acidic deposition compared to low elevation sites within the region. Since the implementation of the Clean Air Act Amendment in the 1990s, concentrations of acidic anions in rainfall have declined. However, some high elevation streams continue to show signs of chronic to episodic acidity, where acid neutralizing capacity (ANC) ranges from 0 to 20 μeq L−1. We studied three 3rd order watersheds (North River in Cherokee National Forest, Santeetlah Creek in Nantahala National Forest, and North Fork of the French Broad in Pisgah National Forest) and selected four to six 1st order catchments within each watershed to represent a gradient in elevation (849–1526 m) and a range in acidic stream ANC values (11–50 μeq L−1). Our objectives were to (1) identify biotic, physical and chemical catchment parameters that could be used as indices of stream ANC, pH and Ca:Al molar ratios and (2) estimate the lime required to restore catchments from the effects of excess acidity and increase base cation availability. We quantified each catchment’s biotic, physical, and chemical characteristics and collected stream, O-horizon, and mineral soil samples for chemical analysis seasonally for one year. Using repeated measures analysis, we examined variability in stream chemistry and catchment characteristics; we used a nested split-plot design to identify catchment characteristics that were correlated with stream chemistry. Watersheds differed significantly and the catchments sampled provided a wide range of stream chemical, biotic, physical and chemical characteristics. Variability in stream ANC, pH, and Ca:Al molar ratio were significantly correlated with catchment vegetation characteristics (basal area, tree height, and tree diameter) as well as O-horizon nitrogen and aluminum concentrations. Total soil carbon and calcium (an indicator of parent material), were significant covariates for stream ANC, pH and Ca:Al molar ratios. Lime requirement estimates did not differ among watersheds but this data will help select catchments for future restoration and lime application studies. Not surprisingly, this work found many vegetation and chemical characteristics that were useful indicators of stream acidity. However, some expected relationships such as concentrations of mineral soil extractable Ca and SO4 were not significant. This suggests that an extensive test of these indicators across the southern Appalachians will be required to identify high elevation forested catchments that would benefit from restoration activities.

In mountainous areas such as the southern Appalachians USA, riparian zones are difficult to define. Vegetation is a commonly used riparian indicator and plays a key role in protecting water resources, but adequate knowledge of floristic responses to riparian disturbances is lacking. Our objective was to quantify changes in stand-level floristic diversity of riparian plant communities before (2004) and two, three, and seven years after shelterwood harvest using highlead cable-yarding and with differing no-cut buffer widths of 0 m, 10 m, and 30 m distance from the stream edge. An unharvested reference stand was also studied for comparison. We examined: (1) differences among treatment sites using a mixed linear model with repeated measures; (2) multivariate relationships between ground-layer species composition and environmental variables (soil water content, light transmittance, tree basal area, shrub density, and distance from stream) using nonmetric multidimensional scaling; and (3) changes in species composition over time using a multi-response permutation procedure. We hypothesized that vegetation responses (i.e., changes in density, species composition, and diversity across the hillslope) will be greatest on harvest sites with an intermediate buffer width (10-m buffer) compared to more extreme (0-m buffer) and less extreme (30-m buffer and no-harvest reference) disturbance intensities. Harvesting initially reduced overstory density and basal area by 83% and 65%, respectively, in the 0-m buffer site; reduced by 50% and 74% in the 10-m buffer site; and reduced by 45% and 29% in the 30-m buffer site. Both the 0-m and 10-m buffer sites showed increased incident light variability across the hillslope after harvesting; whereas, there was no change in the 30-m and reference sites over time. We found significant changes in midstory and ground-layer vegetation in response to harvesting with the greatest responses on the 10-m buffer site, supporting our hypotheses that responses will be greatest on sites with intermediate disturbance. Ground-layer species composition differed significantly over time in the 0-m buffer and 10-m buffer sites (both P P > 0.100). Average compositional dissimilarity increased after seven years, indicating greater within-stand heterogeneity (species diversity) after harvesting. These vegetation recovery patterns provide useful information for evaluating management options in riparian zones in the southern Appalachians.

The pace of environmental and socioeconomic change over the past 100 years has been rapid. Changes in fire regimes, climate, and land use have shaped the structure and function of most forest ecosystems, including oak (Quercus spp. L.) forests in the eastern United States. New stressors such as air pollution and invasive species have contributed to and interacted with climate and fire to alter current forest conditions. While changing fire regimes have altered species composition of the current forest, oak regeneration is constrained by many factors that may affect future forests. Over the remainder of the twenty-first century, an accelerating pace of climate and socioeconomic changes will influence the future range of variation in Eastern oak forests. Some of these impacts will be direct, such as changes in tree growth rates, while other impacts will be indirect, such as new disturbance regimes. While it is likely that fire will be important in shaping oak forests in the twenty-first century, it is less clear exactly what that role will be. For example, it is uncertain whether our current scientific knowledge on the use of prescribed fire in oak forests will be applicable under novel climate and changing socioeconomic conditions. We propose that the combination of climate change, wildfire, and other disturbances will create stand conditions that favor oaks with or without management. However, management intervention (e.g., prescribed fire, thinning, or a combination) could reduce wildfire hazard, particularly in the wildland-urban interface, and create more desirable stand conditions that are resilient to future stressors such as changing precipitation patterns and warmer temperatures.

Climate change and forest disturbances are threatening the ability of forested mountain watersheds to provide the clean, reliable, and abundant fresh water necessary to support aquatic ecosystems and a growing human population. Here, we used 76 years of water yield, climate, and field plot vegetation measurements in six unmanaged, reference watersheds in the southern Appalachian Mountains of North Carolina, USA to determine whether water yield has changed over time, and to examine and attribute the causal mechanisms of change. We found that annual water yield increased in some watersheds from 1938 to the mid-1970s by as much as 55%, but this was followed by decreases up to 22% by 2013. Changes in forest evapotranspiration were consistent with, but opposite in direction to the changes in water yield, with decreases in evapotranspiration up to 31% by the mid-1970s followed by increases up to 29% until 2013. Vegetation survey data showed commensurate reductions in forest basal area until the mid-1970s and increases since that time accompanied by a shift in dominance from xerophytic oak and hickory species to several mesophytic species (i.e., mesophication) that use relatively more water. These changes in forest structure and species composition may have decreased water yield by as much as 18% in a given year since the mid-1970s after accounting for climate. Our results suggest that changes in climate and forest structure and species composition in unmanaged forests brought about by disturbance and natural community dynamics over time can result in large changes in water supply.

Angiotensin II (AngII) has a crucial role in cardiovascular pathologies, including endothelial inflammation and premature vascular aging. However, the precise molecular mechanism underlying aging-related endothelial inflammation induced by AngII remains elusive. Here, we have tested a hypothesis in cultured rat aortic endothelial cells (ECs) that the removal of AngII-induced senescent cells, preservation of proteostasis, or inhibition of mitochondrial fission attenuates the pro-inflammatory EC phenotype. AngII stimulation in ECs resulted in cellular senescence assessed by senescence-associated β galactosidase activity. The number of β galactosidase-positive ECs induced by AngII was attenuated by treatment with a senolytic drug ABT737 or the chemical chaperone 4-phenylbutyrate. Monocyte adhesion assay revealed that the pro-inflammatory phenotype in ECs induced by AngII was alleviated by these treatments. AngII stimulation also increased mitochondrial fission in ECs, which was mitigated by mitochondrial division inhibitor-1. Pretreatment with mitochondrial division inhibitor-1 attenuated AngII-induced senescence and monocyte adhesion in ECs. These findings suggest that mitochondrial fission and endoplasmic reticulum stress have causative roles in endothelial senescence-associated inflammatory phenotype induced by AngII exposure, thus providing potential therapeutic targets in age-related cardiovascular diseases.

Southern Appalachian riparian forests have undergone changes in composition and function from invasive pathogens and pests. Castanea dentata mortality in the 1930s from chestnut blight (Cryphonectria parasitica) and Tsuga canadensis mortality in the 2000s from the hemlock woolly adelgid (Adelges tsugae) have led to the expansion and increased growth of Rhododendron maximum, an evergreen subcanopy shrub. A better understanding of seed bank characteristics and the various abiotic and biotic factors that affect the seed bank may be useful in determining the restoration potential of forest communities following invasion‐related disturbances. We compared the seed bank of two deciduous forest types: hardwood forests with a dense R. maximum subcanopy (hereafter, RR) and hardwood forests without R. maximum (hereafter, HWD). We evaluated numerous microenvironmental variables through principal component analysis (PCA) and correlated the derived PCA axes scores to seed bank density and richness across forest types. We found that seed bank density was comparable between the forests types; however, seed bank richness was much lower in RR than HWD and the species composition was dissimilar between forest types. Twenty‐eight of 64 (44%) species in the seed bank of HWD were not found in the seed bank of RR. Species that were represented in both forest types were often found in contrasting densities. Most notably, seed bank densities of several woody species were considerably higher in RR (85%) than HWD (45%), while herbaceous seed bank density was lower in RR (11%) than HWD (50%). Mineral soil pH, soil nutrient availability, and soil moisture were lower, and organic soil (Oi + Oe + Oa) depth and mass were greater in the RR than HWD forest type. PCA correlations revealed that PCA4 (represented by understory density and Oe + Oa phosphorus and carbon/nitrogen ratio) was negatively correlated with total seed bank density. PCA1 (represented by Oe + Oa cations and phosphorus, understory richness, ground‐layer cover, and mineral soil pH) and PCA4 were positively correlated with total seed bank richness. These results suggest that the soil seed bank will not be the primary mode of recruitment to establish a diverse and herbaceous‐rich community if a RR is present.

Climate change will affect tree species growth and distribution; however, under the same climatic conditions species may differ in their response according to site conditions. We evaluated the climate-driven patterns of growth for six dominant deciduous tree species in the southern Appalachians. We categorized species into two functional groups based on their stomatal regulation and xylem architecture: isohydric, diffuse porous and anisohydric, ring porous. We hypothesized that within the same climatic regime: (i) species-specific differences in growth will be conditional on topographically mediated soil moisture availability; (ii) in extreme drought years, functional groups will have markedly different growth responses; and (iii) multiple hydroclimate variables will have direct and indirect effects on growth for each functional group. We used standardized tree-ring chronologies to examine growth of diffuse-porous (Acer, Liriodendron, and Betula) and ring-porous (Quercus) species vs. on-site climatic data from 1935 to 2003. Quercus species growing on upslope sites had higher basal area increment (BAI) than Quercus species growing on mesic, cove sites; whereas, Acer and Liriodendron had lower BAI on upslope compared to cove sites. Diffuse-porous species were more sensitive to climate than ring porous, especially during extreme drought years. Across functional groups, radial growth was more sensitive to precipitation distribution, such as small storms and dry spell length (DSL), rather than the total amount of precipitation. Based on structural equation modeling, diffuse-porous species on upslope sites were the most sensitive to multiple hydroclimate variables (r(2) = 0.46), while ring-porous species on upslope sites were the least sensitive (r(2) = 0.32). Spring precipitation, vapor pressure deficit, and summer storms had direct effects on summer AET/P, and summer AET/P, growing season small storms and DSL partially explained growth. Decreasing numbers of small storms and extending the days between rainfall events will result in significant growth reduction, even in regions with relatively high total annual rainfall.

The mechanisms by which angiotensin II (AngII) elevates blood pressure and enhances end-organ damage seem to be distinct. However, the signal transduction cascade by which AngII specifically mediates vascular remodeling such as medial hypertrophy and perivascular fibrosis remains incomplete. We have previously shown that AngII-induced epidermal growth factor receptor (EGFR) transactivation is mediated by disintegrin and metalloproteinase domain 17 (ADAM17), and that this signaling is required for vascular smooth muscle cell hypertrophy but not for contractile signaling in response to AngII. Recent studies have implicated endoplasmic reticulum (ER) stress in hypertension. Interestingly, EGFR is capable of inducing ER stress. The aim of this study was to test the hypothesis that activation of EGFR and ER stress are critical components required for vascular remodeling but not hypertension induced by AngII. Mice were infused with AngII for 2 weeks with or without treatment of EGFR inhibitor, erlotinib, or ER chaperone, 4-phenylbutyrate. AngII infusion induced vascular medial hypertrophy in the heart, kidney and aorta, and perivascular fibrosis in heart and kidney, cardiac hypertrophy, and hypertension. Treatment with erlotinib as well as 4-phenylbutyrate attenuated vascular remodeling and cardiac hypertrophy but not hypertension. In addition, AngII infusion enhanced ADAM17 expression, EGFR activation, and ER/oxidative stress in the vasculature, which were diminished in both erlotinib-treated and 4-phenylbutyrate–treated mice. ADAM17 induction and EGFR activation by AngII in vascular cells were also prevented by inhibition of EGFR or ER stress. In conclusion, AngII induces vascular remodeling by EGFR activation and ER stress via a signaling mechanism involving ADAM17 induction independent of hypertension.

This chapter provides information on how to culture primary rat vascular smooth muscle cells and how to induce cellular changes similar to those associated with angiotensin II activation in vivo. We describe how to assess the cellular changes by determining cell size with an automated coulter cell counter to measure cell volume. In addition, we describe a method to assess total protein content. Finally, we describe a standard technique to quantify angiotensin II-induced pro-fibrotic response using the Chondrex Sirius Red Total Collagen Detection Kit.

Studying signal transduction in hypertension model systems in vitro will include several steps: (1) develop the cell culture model and induce hypertensive changes, (2) observe kinase activation, (3) manipulate signal transduction pathways, and (4) observe physiologic outputs. This chapter will provide the reader with overviews of the techniques our lab uses to inhibit signaling pathways with inhibitory RNAs and the outputs we use to monitor the effects.

Studying signal transduction in hypertension model systems in vitro will include several steps: (1) develop the cell culture model and induce hypertensive changes, (2) observe kinase activation, (3) manipulate signal transduction pathways, and (4) observe physiologic outputs. This chapter will provide the reader with overviews of the techniques our lab uses to inhibit signaling pathways with inhibitory RNAs and the outputs we use to monitor the effects.

This chapter provides information on how to culture primary rat vascular smooth muscle cells and how to induce cellular changes similar to those associated with angiotensin II activation in vivo. We describe how to assess the cellular changes by determining cell size with an automated coulter cell counter to measure cell volume. In addition, we describe a method to assess total protein content. Finally, we describe a standard technique to quantify angiotensin II-induced pro-fibrotic response using the Chondrex Sirius Red Total Collagen Detection Kit.

Using an in vivo model system to study signal transduction will include several steps: (1) induce hypertension in the animal, (2) manipulate kinase activation and signal transduction pathways as desired, and (3) observe physiologic outputs. This chapter provides the reader with overviews of the techniques our lab uses to manipulate signal transduction pathways and determine the effects on hypertension.

The importance of the herbaceous layer in regulating ecosystem processes in deciduous forests is generally unknown. We use a manipulative study in a rich, mesophytic cove forest in the southern Appalachians to test the following hypotheses: (i) the herbaceous functional group (HFG) in mesophytic coves accelerates carbon and nutrient cycling, (ii) high litter quality input and rapid nutrient turnover associated with HFG will have a positive effect on overstory tree growth, and (iii) the HFG regulates tree regeneration with negative effects on seedling establishment due to competition for resources. We established treatment plots in a mesic, cove-hardwoods forest and removed the herbaceous flora (HR, removed twice per year) or added herbaceous organic material (OMA, once per year) for comparison to a no removal (NR) reference for a total of 14 years. The OMA treatment stimulated soil N-mineralization and increased litterfall mass and N content. OMA N-mineralization rates were more than two times greater than both the NR and HR treatments; however, we did not detect significant differences in soil CO2 efflux among treatments. Higher overstory litterfall mass and N in the OMA treatment plots indicated that overstory trees were benefiting from the enhanced soil N-mineralization. Higher overstory leaf mass and N suggests an important linkage between HR and aboveground net primary production even though this did not translate into greater tree basal area increment. We found an increase in regeneration of all tree species with HFG removal, and the response was particularly evident for Acer rubrum seedlings.

Fire has a profound, though paradoxical influence on landscapes of the southeastern U.S.; it simultaneously maintains native biodiversity and ecosystem processes but also threatens silvicultural resources and human landscapes. Furthermore, since the majority of the southern landscape is heavily influenced by human activities, contemporary fire regimes are human managed disturbances within extant firedependent ecosystems. Though there is considerable uncertainty in climate projections for the southeastern U.S., climate change will likely impact both prescribed fire and wildfire. In this review, we synthesize climate change-fire interactions, discuss the impacts of uncertainty in a human-dominated landscape, and illuminate how both climate change projections and their uncertainties might impact our ability to manage forests in the Southeast. We define the Southeast region as consisting of the Gulf Coastal Plain, Lower Atlantic Coastal Plain, Piedmont and southern Appalachians and associated subregions. This region has the greatest area burned by prescribed fire, the highest number of wildfires in the continental U.S. and contains globally significant hotspots of biodiversity, much of which is dependent on frequent fire. The use of prescribed fire as a management tool depends on a suite of weather and fuel conditions which are affected by climate. Over the next five decades, general circulation models (GCMs) consistently predict air temperature to increase by 1.5–3 C in the Southeast. Precipitation forecasts are more uncertain with respect to the mean; but, most models predict an increase in precipitation variability. Increases in the likelihood of severe droughts may increase wildfire occurrence while simultaneously limiting the implementation of prescribed burning by restricting the number of days within current prescription guidelines. While the Southeast has among the highest potential for C storage and sequestration, a reduction in C sequestration capacity due to increasing disturbances such as drought, insect infestations, hurricanes and fire, is possible. The potential for long-term shifts in forest composition from climate-altered fire regimes if coupled with an increased potential for wildfire occurrence could reduce quality and quantity of water released from forests at times when demand for high quality water will intensify for human use. Furthermore, any reduction in prescribed burning is likely to result in decreased biological diversity, particularly in the Coastal Plain, a global hotspot of biodiversity. Lastly, more future area burned by wildfire rather than prescribed fire has the potential to negatively influence regional air quality. Mitigating the negative effects of climate change-fire interactions would require actively exploiting favorable seasonal and inter-annual climate windows. Monitoring the type conversions of agricultural and fiber production forest will be critical for long-term projections of fire risk and watershed impacts of altered fire regimes.

Understanding the patterns of past disturbance allows further insight into the complex composition, structure, and function of current and future forests, which is increasingly important in a world where disturbance characteristics are changing. Our objectives were to define disturbance causes, rates (percent disturbance per decade), magnitudes and frequency (time since last disturbance) for both secondary and old-growth mixed-oak stands, and to determine if all mixed oak stands experience similar disturbance history. The study was located in two southern Appalachian forests in western North Carolina, USA: Coweeta Hydrologic Laboratory, a 2,185 ha experimental forest with some history of harvesting, and the Joyce Kilmer Wilderness, a 6,805 ha old-growth forest with no known harvesting. We used dendroecological techniques to evaluate the disturbance histories and create chronologies of these mixed-oak forests. Average decadal disturbance rates ranged from 4.3% to 13.8%, similar to rates common in ...

Although AngII (angiotensin II) and its receptor AT1R (AngII type 1 receptor) have been implicated in AAA (abdominal aortic aneurysm) formation, the proximal signalling events primarily responsible for AAA formation remain uncertain. Caveolae are cholesterol-rich membrane microdomains that serve as a signalling platform to facilitate the temporal and spatial localization of signal transduction events, including those stimulated by AngII. Cav1 (caveolin 1)-enriched caveolae in vascular smooth muscle cells mediate ADAM17 (a disintegrin and metalloproteinase 17)-dependent EGFR (epidermal growth factor receptor) transactivation, which is linked to vascular remodelling induced by AngII. In the present study, we have tested our hypothesis that Cav1 plays a critical role for the development of AAA at least in part via its specific alteration of AngII signalling within caveolae. Cav1−/− mice and the control wild-type mice were co-infused with AngII and β-aminopropionitrile to induce AAA. We found that Cav1−/− mice with the co-infusion did not develop AAA compared with control mice in spite of hypertension. We found an increased expression of ADAM17 and enhanced phosphorylation of EGFR in AAA. These events were markedly attenuated in Cav1−/− aortas with the co-infusion. Furthermore, aortas from Cav1−/− mice with the co-infusion showed less endoplasmic reticulum stress, oxidative stress and inflammatory responses compared with aortas from control mice. Cav1 silencing in cultured vascular smooth muscle cells prevented AngII-induced ADAM17 induction and activation. In conclusion, Cav1 appears to play a critical role in the formation of AAA and associated endoplasmic reticulum/oxidative stress, presumably through the regulation of caveolae compartmentalized signals induced by AngII.

We synthesized the current information on mesophytic cove forests in the southern Appalachians, assessed the range of variation in herb species composition and diversity in stands with different disturbance histories and environmental conditions, identified key knowledge gaps, and suggested approaches to fill these knowledge gaps. The purpose of this synthesis was to provide information to forest managers to help make decisions about conservation assessments and strategies for rich cove forests in the southern Appalachians. An important finding is that no single study or data set can provide conclusive evidence or clear management strategies. However, an overriding conclusion is that the magnitude of impact and the management actions necessary to restore herbaceous communities are directly proportional to the severity of disturbance, current condition (e.g., presence of Rhododendron), site heterogeneity, and historical land use (e.g., agricultural activity). These factors plus a host of other str...

The Max-interacting protein Mnt is a transcriptional repressor that can antagonize the transcriptional and proliferation-related activities of Myc. Here, we tested the hypothesis that Mnt is a negative regulator of pathological vascular remodeling.Adenovirus encoding Mnt or control GFP was infected to cultured rat vascular smooth muscle cells (VSMC) and carotid arteries after a balloon angioplasty.In VSMC, adenoviral gene transfer of Mnt suppressed angiotensin II-induced protein expression of early growth response protein-1 (Egr1) and its promoter activation. Mnt adenovirus did not interfere with upstream signaling of angiotensin II. Angiotensin II-induced protein accumulation in VSMC was inhibited by Mnt adenovirus. Mnt adenovirus also inhibited platelet-derived growth factor-induced VSMC proliferation. Moreover, Mnt adenovirus prevented neointima formation in response to arterial injury. The adenoviral Mnt gene transfer also prevented Egr1 induction in neointima.These data identify Mnt as a previously unrecognized negative regulator of pathological vascular remodeling.