The recurring role of site challenges assumptions about regeneration under selection systems in northern hardwoods

In naturally regenerated managed forests, silvicultural methods leverage timing and intensity of harvesting activities to align with species-specific reproduction mechanisms. With contemporary emphasis on complex stand structure and diverse composition, there is uncertainty in the continued use of timber-oriented management practices in meeting evolving objectives. In the northern hardwood region of North America, selection regeneration systems are assumed to result in homogenization of structure and composition through increasing dominance of Acer saccharum Marsh. Given the coupling of soils and vegetation in northern hardwoods, trends in site conditions that may be more resilient/facilitative to community diversity may be of value to silviculturists. Remote sensing products and inventory records were integrated to assess tree communities across site variables in northern Michigan, USA. Results reveal that composition is stabilized by local landforms and diversity increases with hydrologic catchment area. Time since treatment (0-54 years) appeared negatively correlated with catchment area, suggesting lowlands with high diversity are not managed or harvested infrequently, reflecting equipment access and operational logistics. Broad interpretations of selection regeneration systems may be invalidated by the influence of site conditions not previously accounted for, and results highlight a novel technique to capture the effect of topography on species assemblages.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Incidental Effects of Cut-to-Length Harvest Systems and Residue Management on Populus tremuloides (Michx.) Regeneration and Yield

In Great Lakes aspen forests, clearcutting and whole-tree skidding (WTS) has been the usual practice. Soil disturbances from WTS sometimes lead to regeneration declines in areas of high equipment traffic. Advances in harvesting mechanization have resulted in increased utilization of cut-to-length (CTL) processing equipment, which can reduce site impact through placing harvest residues in operational corridors to spread surface load posed by equipment. While these practices may mitigate soil disturbance, heavy residue loads can decrease aspen regeneration. We sampled soil conditions and regeneration systematically, in transects perpendicular to CTL trails, and compiled metrics of residue loads, regeneration density and height, and soil characteristics to examine potential interactions. Our results indicate that residues are notably greater directly on the equipment trail when compared to adjacent areas, and that these trends correspond with a significant loss in regeneration height and density. Further, we observed a 186% increase in the stand area affected by CTL operations compared to those reported for WTS methods elsewhere. We projected these trends with growth and yield simulations, which suggest a reduction in timber yields at rotation age as compared to stands without explicit CTL equipment trails. These patterns may have unintended influences on effective stand regeneration and threaten short-term productivity and future yields. The impact of harvesting equipment and residues on edaphic conditions and recovering vegetation is likely site and cover type specific

Vegetation response to logging residue removals in Great Lakes aspen forests: Long-term trends under operational management

© 2016 Elsevier B.V. Conservation of vegetation diversity has been integrated into forest management regimes as stands with high levels of diversity have been shown to possess enhanced ecological services and resilience to disturbance. Effects of intensified forest management and harvesting practices such as logging residue removals for bioenergy on vegetation diversity and community dynamics has been largely overlooked, however. We examined forest vegetation communities under residue removal treatments along a unique 40-yr chronosequence of commercial aspen (Populus spp.) in Upper Michigan to examine potential impacts on species abundance metrics, community structure, and heterogeneity. We hypothesized that residue removals would negatively impact species diversity of both the over- and understory communities, and that these patterns would occur in parallel with shifts in community structure of both strata. Our results show no effect of residue treatment on overstory tree communities; however we did observe an increase in understory species richness, diversity, and evenness in stands where residues were harvested. The understory community structure was correlated with soil total nitrogen levels, yet this pattern was independent of harvest residue treatment. In addition, we observed higher levels of variance in species representation of the understory community when residues were removed, but did not detect any difference in community organization. Overall, our results suggest that the removal of harvest residues can increase variability in vegetation community relationships. We believe that these patterns can be attributed to increased site disturbance and soil scarification through additional equipment trafficking and subsequent changes in micro environmental conditions when residues are harvested

Abstract 395: Transient Inactivation of Retinoblastoma Induces De-Novo Cardiomyogenesis From Human Myocardial Progenitors but Not Pre-Existing Cardiomyocyte Replication

Introduction: Activation of cardiac cell cycle re-entry is considered the primary therapeutic strategy for cardiomyocyte (CM) regeneration. However, the role of cardiac cell-cycle control in cardiomyogenesis remains elusive. Here, we combined RNA interference and stem cell modeling to investigate the role of Retinoblastoma (RB) in human cardiomyogenesis. Hypothesis: RB regulates proliferation and differentiation of cardiac progenitors (CPCs) but not CM replication. Methods: H9 human embryonic stem cells (hESCs) stably expressing tetracycline (tet)-inducible shRNAs against RB (hESCshRB) or hemagglutinin-tagged RB (hESCHA-RB) were tet-induced at selected time-points during or after CM differentiation. Results: Analysis of ser-608 illustrated stage-specific differences in the degree of RB inactivation during normal hESCs-cardiogenesis. Transient shRB knockdown in hESCshRB-derived embryoid bodies (EBs) during the CPC-stage (EB-days 5-8), significantly upregulated GATA4, ISL1, CTNNI, and cKit transcription (p<0.05), while increasing the yield of beating EBs by 2.4-fold (n=6/group, p<0.0001 vs. vehicle). Gene-expression arrays of 22 RB-related genes, illustrated that shRB-knockdown upregulated CCND1, CCND2, CCND3, and CDK4, CDK6 (p<0.05), followed by a 3.6-fold increase in E2F3 (p<0.05) expression. Moreover, expression of p107 and p130, p27, p57, ARF and CDKN3 were also significantly increased (p<0.05), whereas TP53 and MDM2 remained unchanged. Ectopic HA-RB in CPCs did not significantly affect cardiogenesis (n=18). Conversely, shRB knockdown in EB-day 60-derived CMs (n=15) did not stimulate cell cycle re-entry, as assessed by analysis of EdU incorporation and Aurora-B kinase (AurB). Remarkably, co-culture of hESCHA-RB-derived CMs with adult cardiac (CSCs) and/or mesenchymal (MSCs) stem cells (n=15/group), increased cell-cycle re-entry ~2.8-fold, assessed by ser-10 Histone H3 (p=0.0002) and AurB (p<0.0001). Conclusions: These findings suggest that RB regulates proliferation and differentiation of human CPCs in a cell-autonomous manner, via a CCND-CDK4/6-E2F3 mechanism. Conversely, CM replication may be enhanced via cell-cell interactions with MSCs and/or CSCs, but not cell-autonomously via RB inactivation

Allogeneic Mesenchymal Stem Cells Restore Endothelial Function in Heart Failure by Stimulating Endothelial Progenitor Cells

Background: Endothelial dysfunction, characterized by diminished endothelial progenitor cell (EPC) function and flow-mediated vasodilation (FMD), is a clinically significant feature of heart failure (HF). Mesenchymal stem cells (MSCs), which have pro-angiogenic properties, have the potential to restore endothelial function. Accordingly, we tested the hypothesis that MSCs increase EPC function and restore flow-mediated vasodilation (FMD). Methods: Idiopathic dilated and ischemic cardiomyopathy patients were randomly assigned to receive autologous (n = 7) or allogeneic (n = 15) MSCs. We assessed EPC-colony forming units (EPC-CFUs), FMD, and circulating levels of vascular endothelial growth factor (VEGF) in patients before and three months after MSC transendocardial injection (n = 22) and in healthy controls (n = 10). Findings: EPC-colony forming units (CFUs) were markedly reduced in HF compared to healthy controls (4 ± 3 vs. 25 ± 16 CFUs, P < 0.0001). Similarly, FMD% was impaired in HF (5.6 ± 3.2% vs. 9.0 ± 3.3%, P = 0.01). Allogeneic, but not autologous, MSCs improved endothelial function three months after treatment (Δ10 ± 5 vs. Δ1 ± 3 CFUs, P = 0.0067; Δ3.7 ± 3% vs. Δ-0.46 ± 3% FMD, P = 0.005). Patients who received allogeneic MSCs had a reduction in serum VEGF levels three months after treatment, while patients who received autologous MSCs had an increase (P = 0.0012), and these changes correlated with the change in EPC-CFUs (P < 0.0001). Lastly, human umbilical vein endothelial cells (HUVECs) with impaired vasculogenesis due to pharmacologic nitric oxide synthase inhibition, were rescued by allogeneic MSC conditioned medium (P = 0.006). Interpretation: These findings reveal a novel mechanism whereby allogeneic, but not autologous, MSC administration results in the proliferation of functional EPCs and improvement in vascular reactivity, which in turn restores endothelial function towards normal in patients with HF. These findings have significant clinical and biological implications for the use of MSCs in HF and other disorders associated with endothelial dysfunction

Abstract 214: Reduction of Scar Tissue after GHRH-A Treatment in a Swine Model of Sub-acute Ischemic Cardiomyopathy

Background: Growth hormone-releasing hormone receptor agonists (GHRH-A) stimulate cardiac repair following myocardial infarction (MI) through the activation of the GHRH signaling pathway within the heart. We tested the hypothesis that the administration of GHRH-A prevents ventricular remodeling in a swine sub-acute MI model. Methods: Twelve female Yorkshire swine (25-30 Kg) underwent transient occlusion of the LAD coronary artery (MI). Two-weeks post-MI, swine were randomized to receive injections of either 30 μg/Kg GHRH-A (MR-409) (GHRH-A group; n=6) or vehicle (placebo group; n=6). Cardiac MRI, pressure volume loops and measures of endothelial function were obtained at multiple time points. Infarct-, border- and remote- (non-infarcted) zones were assessed by immunohistochemistry for the growth hormone-releasing hormone receptor (GHRHR). Results: Four-weeks of GHRH-A treatment resulted in reduced scar mass (GHRH-A group: –21.9±6.42%; p=0.02; placebo group: 10.9±5.88%; p=0.25; Two-way ANOVA; p=0.003), and reduced scar size (percent of left ventricle mass) (GHRH-A group: –38.38±4.63; p=0.0002; placebo group: –14.56± 6.92; p=0.16; Two-way ANOVA; p=0.02). Moreover, peripheral endothelial function was significantly increased compared to baseline values in the GHRH-A group (paired t-test; p=0.006) but not in the placebo group (p=0.99). Unlike in rats, this reduced infarct size in swine was not accompanied by improved cardiac function as measured by serial hemodynamic pressure-volume analysis. GHRH receptors were abundant in cardiac tissue, with a greater density in the border zone of the GHRH-A group compared to the placebo group. These data support the concept of direct post-infarction activation of cardiac signal transduction, and of enhancing this activation with systemic treatment by GHRH. Conclusions: Daily subcutaneous administration of GHRH-A is feasible and safe in female swine. Furthermore, GHRH-A therapy significantly reduced infarct size and increased endothelial function, suggesting that a local activation of the GHRH pathway leads to the regenerative process and preservation of peripheral endothelial function

Abstract 140: Effect of Transendocardial Autologous Cardiac Stem Cells and Bone Marrow Mesenchymal Stem Cells to Reduce Infarct Size and Restore Cardiac Function in a Heart Failure Swine Model

Background: A cell combination of human mesenchymal stem cells (MSCs) and c-kit+ cardiac stem cells (CSCs) improves left ventricular (LV) performance to a greater degree than MSCs alone in post myocardial infarction swine. To advance the development of cell combination therapy, we administered autologous swine cells, and tested the hypothesis that transendocardial autologous CSCs/MSCs produces greater improvement of performance than MSCs in a rigorous model of heart failure due to post infarct LV remodeling. Methods: Gottingen mini-swine (n=28) underwent LAD coronary artery occlusion followed by reperfusion, and allowed to undergo LV remodeling for 90 days. Autologous MSCs were amplified from bone marrow and CSCs from right ventricular biopsies in each swine, and injections of either CSC/MSC combo (1M/200M, n=7), MSCs (200M, n=7), or placebo (Plasmalyte, n=6) were injected to the infarct-border zone via the NOGA system. Cardiac MRI and pressure volume loops were obtained before and after therapy. Results: Both cell groups had substantially reduced scar size (Combo –37.2.9± 5.4% vs MSCs –38.8±7.5% vs placebo −7.2±6.3, P=0.0001) and increased viable tissue (Combo +30.9±7% vs MSCs +41.8±10.5% vs placebo +7.7±4.5, P<0.0001) relative to placebo. Ejection Fraction (EF) improved only in the Combo group (Combo +7.0±2.8 vs MSCs +3.4±1.3 vs placebo +1.2±1.6 EF units, P=0.04). Accompanying this EF restoration was a substantial improvement in the Combo group in stroke volume (Combo +47.2±11.1% vs MSCs +32.6±12.0% vs placebo +10.8±4.5, P<0.0001), cardiac output (Combo +35.9±7.6% vs MSCs 41.9±26.5% vs placebo −16.4±6.6%, P=0.01) and diastolic strain rate (Combo +18.9±8.6% vs MSCs 14.0±8.8% vs placebo −14.9±9.5%, P=0.03). Conclusions: Combination cell therapy and MSCs alone dramatically reduce scar size in a swine model of chronic ischemic cardiomyopathy. In contrast, combination therapy has much greater impact on functional recovery, increasing EF to [near normal] levels. These findings illustrate that interactions between ckit+ CSCs and MSCs result in substantial enhancement in cardiac performance, establish the safety of autologous cell combination strategies, and support the development of an advanced second generation cell therapeutic product

Randomized Comparison of Allogeneic Versus Autologous Mesenchymal Stem Cells for Nonischemic Dilated Cardiomyopathy

BACKGROUND: While human mesenchymal stem cells (hMSCs) have been tested in ischemic cardiomyopathy, few studies exist in chronic non-ischemic dilated cardiomyopathy (NIDCM). OBJECTIVES: The POSEIDON-DCM trial is a randomized comparison of safety and efficacy of autologous (auto) vs. allogeneic (allo) bone marrow-derived hMSCs in NIDCM. METHODS: Thirty-seven patients were randomized to either allo- or auto-hMSCs in a 1:1 ratio. Patients were recruited between December 2011 and July 2015 at the University of Miami Hospital. Patients (age: 55.8 ± 11.2; 32% female) received hMSCs (100 million) by transendocardial stem cell injection (TESI) in ten left ventricular sites by NOGA Catheter. Treated patients were evaluated at baseline, 30 days, 3-, 6-, and 12-months for safety: serious adverse events (SAE), and efficacy endpoints: Ejection Fraction (EF), Minnesota Living with Heart Failure Questionnaire (MLHFQ), Six Minute Walk Test (6MWT), MACE, and immune-biomarkers. This trial is registered with ClinicalTrials.gov, #NCT01392625. RESULTS: There were no 30-day treatment-emergent (TE)-SAEs. 12-month SAE incidence was 28.2% (95% CI: 12.8, 55.1) in allo, and 63.5% (95% CI: 40.8, 85.7; p=0.1004) in auto. One allo-group patient developed an elevated donor specific cPRA. EF increased in allo by 8.0 units (95% Cl: 2.8, 13.2; p=0.004), and in auto: 5.4 units (95% Cl: −1.4, 12.1; p=0.116, allo vs. auto p=0.4887). 6MWT increased for allo: 37.0 meters (95% Cl: 2.0 to 72.0; p=0.04), but not auto: 7.3 meters (95% Cl: −47.8, 33.3; p=0.71, auto vs. allo p=0.0168). MLHFQ score decreased in allo (p=0.0022), and auto (p=0.463; p=0.172). The MACE rate was lower in allo vs. auto (p=0.0186). Tumor necrosis factor alpha (TNF-α) decreased (p=0.0001 for each), to a greater extent in allo vs. auto at six-months (p=0.05). CONCLUSION: These findings demonstrate safety and support greater, clinically meaningful efficacy of allo-hMSC vs. auto-hMSC in NIDCM patients. Pivotal trials of allo-hMSCs are warranted based on these results