Dual modulation of cell survival and cell death by beta(2)-adrenergic signaling in adult mouse cardiac myocytes.

The goal of this study was to determine whether beta(1)-adrenergic receptor (AR) and beta(2)-AR differ in regulating cardiomyocyte survival and apoptosis and, if so, to explore underlying mechanisms. One potential mechanism is that cardiac beta(2)-AR can activate both G(s) and G(i) proteins, whereas cardiac beta(1)-AR couples only to G(s). To avoid complicated crosstalk between beta-AR subtypes, we expressed beta(1)-AR or beta(2)-AR individually in adult beta(1)/beta(2)-AR double knockout mouse cardiac myocytes by using adenoviral gene transfer. Stimulation of beta(1)-AR, but not beta(2)-AR, markedly induced myocyte apoptosis, as indicated by increased terminal deoxynucleotidyltransferase-mediated UTP end labeling or Hoechst staining positive cells and DNA fragmentation. In contrast, beta(2)-AR (but not beta(1)-AR) stimulation elevated the activity of Akt, a powerful survival signal; this effect was fully abolished by inhibiting G(i), G(beta gamma), or phosphoinositide 3 kinase (PI3K) with pertussis toxin, beta ARK-ct (a peptide inhibitor of G(beta gamma)), or LY294002, respectively. This indicates that beta(2)-AR activates Akt via a G(i)-G(beta gamma)-PI3K pathway. More importantly, inhibition of the G(i)-G(beta gamma)-PI3K-Akt pathway converts beta(2)-AR signaling from survival to apoptotic. Thus, stimulation of a single class of receptors, beta(2)-ARs, elicits concurrent apoptotic and survival signals in cardiac myocytes. The survival effect appears to predominate and is mediated by the G(i)-G(beta gamma)-PI3K-Akt signaling pathway.

Preservation of myocardial beta-adrenergic receptor signaling delays the development of heart failure after myocardial infarction.

When the heart fails, there is often a constellation of biochemical alterations of the beta-adrenergic receptor (betaAR) signaling system, leading to the loss of cardiac inotropic reserve. betaAR down-regulation and functional uncoupling are mediated through enhanced activity of the betaAR kinase (betaARK1), the expression of which is increased in ischemic and failing myocardium. These changes are widely viewed as representing an adaptive mechanism, which protects the heart against chronic activation. In this study, we demonstrate, using in vivo intracoronary adenoviral-mediated gene delivery of a peptide inhibitor of betaARK1 (betaARKct), that the desensitization and down-regulation of betaARs seen in the failing heart may actually be maladaptive. In a rabbit model of heart failure induced by myocardial infarction, which recapitulates the biochemical betaAR abnormalities seen in human heart failure, delivery of the betaARKct transgene at the time of myocardial infarction prevents the rise in betaARK1 activity and expression and thereby maintains betaAR density and signaling at normal levels. Rather than leading to deleterious effects, cardiac function is improved, and the development of heart failure is delayed. These results appear to challenge the notion that dampening of betaAR signaling in the failing heart is protective, and they may lead to novel therapeutic strategies to treat heart disease via inhibition of betaARK1 and preservation of myocardial betaAR function.

Combined Gene Therapy and Functional Tissue Engineering for the Treatment of Osteoarthritis

The pathogenesis of osteoarthritis is mediated in part by inflammatory cytokines including interleukin-1 (IL-1), which promote degradation of articular cartilage and prevent human mesenchymal stem cell (hMSC) chondrogenesis. We combined gene therapy and functional tissue engineering to develop engineered cartilage with immunomodulatory properties that allow chondrogenesis in the presence of pathologic levels of IL-1 by inducing overexpression of IL-1 receptor antagonist (IL-1Ra) in hMSCs via scaffold-mediated lentiviral gene delivery. A doxycycline-inducible vector was used to transduce hMSCs in monolayer or within 3D woven PCL scaffolds to enable tunable IL-1Ra production. In the presence of IL-1, IL-1Ra-expressing engineered cartilage produced cartilage-specific extracellular matrix, while resisting IL-1-induced upregulation of matrix metalloproteinases and maintaining mechanical properties similar to native articular cartilage. The ability of functional engineered cartilage to deliver tunable anti-inflammatory cytokines to the joint may enhance the long-term success of therapies for cartilage injuries or osteoarthritis.

Following this, we modified this anti-inflammatory engineered cartilage to incorporate rabbit MSCs and evaluated this therapeutic strategy in a pilot study in vivo in rabbit osteochondral defects. Rabbits were fed a custom doxycycline diet to induce gene expression in engineered cartilage implanted in the joint. Serum and synovial fluid were collected and the levels of doxycycline and inflammatory mediators were measured. Rabbits were euthanized 3 weeks following surgery and tissues were harvested for analysis. We found that doxycycline levels in serum and synovial fluid were too low to induce strong overexpression of hIL-1Ra in the joint and hIL-1Ra was undetectable in synovial fluid via ELISA. Although hIL-1Ra expression in the first few days local to the site of injury may have had a beneficial effect, overall a higher doxycycline dose and more readily transduced cell population would improve application of this therapy.

In addition to the 3D woven PCL scaffold, cartilage-derived matrix scaffolds have recently emerged as a promising option for cartilage tissue engineering. Spatially-defined, biomaterial-mediated lentiviral gene delivery of tunable and inducible morphogenetic transgenes may enable guided differentiation of hMSCs into both cartilage and bone within CDM scaffolds, enhancing the ability of the CDM scaffold to provide chondrogenic cues to hMSCs. In addition to controlled production of anti-inflammatory proteins within the joint, in situ production of chondro- and osteo-inductive factors within tissue-engineered cartilage, bone, or osteochondral tissue may be highly advantageous as it could eliminate the need for extensive in vitro differentiation involving supplementation of culture media with exogenous growth factors. To this end, we have utilized controlled overexpression of transforming growth factor-beta 3 (TGF-β3), bone morphogenetic protein-2 (BMP-2) or a combination of both factors, to induce chondrogenesis, osteogenesis, or both, within CDM hemispheres. We found that TGF-β3 overexpression led to robust chondrogenesis in vitro and BMP-2 overexpression led to mineralization but not accumulation of type I collagen. We also showed the development of a single osteochondral construct by combining tissues overexpressing BMP-2 (hemisphere insert) and TGF-β3 (hollow hemisphere shell) and culturing them together in the same media. Chondrogenic ECM was localized in the TGF-β3-expressing portion and osteogenic ECM was localized in the BMP-2-expressing region. Tissue also formed in the interface between the two pieces, integrating them into a single construct.

Since CDM scaffolds can be enzymatically degraded just like native cartilage, we hypothesized that IL-1 may have an even larger influence on CDM than PCL tissue-engineered constructs. Additionally, anti-inflammatory engineered cartilage implanted in vivo will likely affect cartilage and the underlying bone. There is some evidence that osteogenesis may be enhanced by IL-1 treatment rather than inhibited. To investigate the effects of an inflammatory environment on osteogenesis and chondrogenesis within CDM hemispheres, we evaluated the ability of IL-1Ra-expressing or control constructs to undergo chondrogenesis and osteogenesis in the prescence of IL-1. We found that IL-1 prevented chondrogenesis in CDM hemispheres but did not did not produce discernable effects on osteogenesis in CDM hemispheres. IL-1Ra-expressing CDM hemispheres produced robust cartilage-like ECM and did not upregulate inflammatory mediators during chondrogenic culture in the presence of IL-1.

Salmeterol enhances the cardiac response to gene therapy in Pompe disease.

Enzyme replacement therapy (ERT) with recombinant human (rh) acid α-glucosidase (GAA) has prolonged the survival of patients. However, the paucity of cation-independent mannose-6-phosphate receptor (CI-MPR) in skeletal muscle, where it is needed to take up rhGAA, correlated with a poor response to ERT by muscle in Pompe disease. Clenbuterol, a selective β2 receptor agonist, enhanced the CI-MPR expression in striated muscle through Igf-1 mediated muscle hypertrophy, which correlated with increased CI-MPR (also the Igf-2 receptor) expression. In this study we have evaluated 4 new drugs in GAA knockout (KO) mice in combination with an adeno-associated virus (AAV) vector encoding human GAA, 3 alternative β2 agonists and dehydroepiandrosterone (DHEA). Mice were injected with AAV2/9-CBhGAA (1E+11 vector particles) at a dose that was not effective at clearing glycogen storage from the heart. Heart GAA activity was significantly increased by either salmeterol (p<0.01) or DHEA (p<0.05), in comparison with untreated mice. Furthermore, glycogen content was reduced in the heart by treatment with DHEA (p<0.001), salmeterol (p<0.05), formoterol (p<0.01), or clenbuterol (p<0.01) in combination with the AAV vector, in comparison with untreated GAA-KO mice. Wirehang testing revealed that salmeterol and the AAV vector significantly increased performance, in comparison with the AAV vector alone (p<0.001). Similarly, salmeterol with the vector increased performance significantly more than any of the other drugs. The most effective individual drugs had no significant effect in absence of vector, in comparison with untreated mice. Thus, salmeterol should be further developed as adjunctive therapy in combination with either ERT or gene therapy for Pompe disease.

Extended-release ranolazine: critical evaluation of its use in stable angina.

Coronary heart disease is the major cause of morbidity and mortality throughout the world, and is responsible for approximately one of every six deaths in the US. Angina pectoris is a clinical syndrome characterized by discomfort, typically in the chest, neck, chin, or left arm, induced by physical exertion, emotional stress, or cold, and relieved by rest or nitroglycerin. The main goals of treatment of stable angina pectoris are to improve quality of life by reducing the severity and/or frequency of symptoms, to increase functional capacity, and to improve prognosis. Ranolazine is a recently developed antianginal with unique methods of action. In this paper, we review the pharmacology of ranolazine, clinical trials supporting its approval for clinical use, and studies of its quality of life benefits. We conclude that ranolazine has been shown to be a reasonable and safe option for patients who have refractory ischemic symptoms despite the use of standard medications (for example, nitrates, beta-adrenergic receptor antagonists, and calcium channel antagonists) for treatment of anginal symptoms, and also provides a modestly improved quality of life.

Using Bird Distributions to Assess Extinction Risk and Identify Conservation Priorities in Biodiversity Hotspots

Habitat loss, fragmentation, and degradation threaten the World’s ecosystems and species. These, and other threats, will likely be exacerbated by climate change. Due to a limited budget for conservation, we are forced to prioritize a few areas over others. These places are selected based on their uniqueness and vulnerability. One of the most famous examples is the biodiversity hotspots: areas where large quantities of endemic species meet alarming rates of habitat loss. Most of these places are in the tropics, where species have smaller ranges, diversity is higher, and ecosystems are most threatened.

Species distributions are useful to understand ecological theory and evaluate extinction risk. Small-ranged species, or those endemic to one place, are more vulnerable to extinction than widely distributed species. However, current range maps often overestimate the distribution of species, including areas that are not within the suitable elevation or habitat for a species. Consequently, assessment of extinction risk using these maps could underestimate vulnerability.

In order to be effective in our quest to conserve the World’s most important places we must: 1) Translate global and national priorities into practical local actions, 2) Find synergies between biodiversity conservation and human welfare, 3) Evaluate the different dimensions of threats, in order to design effective conservation measures and prepare for future threats, and 4) Improve the methods used to evaluate species’ extinction risk and prioritize areas for conservation. The purpose of this dissertation is to address these points in Colombia and other global biodiversity hotspots.

In Chapter 2, I identified the global, strategic conservation priorities and then downscaled to practical local actions within the selected priorities in Colombia. I used existing range maps of 171 bird species to identify priority conservation areas that would protect the greatest number of species at risk in Colombia (endemic and small-ranged species). The Western Andes had the highest concentrations of such species—100 in total—but the lowest densities of national parks. I then adjusted the priorities for this region by refining these species ranges by selecting only areas of suitable elevation and remaining habitat. The estimated ranges of these species shrank by 18–100% after accounting for habitat and suitable elevation. Setting conservation priorities on the basis of currently available range maps excluded priority areas in the Western Andes and, by extension, likely elsewhere and for other taxa. By incorporating detailed maps of remaining natural habitats, I made practical recommendations for conservation actions. One recommendation was to restore forest connections to a patch of cloud forest about to become isolated from the main Andes.

For Chapter 3, I identified areas where bird conservation met ecosystem service protection in the Central Andes of Colombia. Inspired by the November 11th (2011) landslide event near Manizales, and the current poor results of Colombia’s Article 111 of Law 99 of 1993 as a conservation measure in this country, I set out to prioritize conservation and restoration areas where landslide prevention would complement bird conservation in the Central Andes. This area is one of the most biodiverse places on Earth, but also one of the most threatened. Using the case of the Rio Blanco Reserve, near Manizales, I identified areas for conservation where endemic and small-range bird diversity was high, and where landslide risk was also high. I further prioritized restoration areas by overlapping these conservation priorities with a forest cover map. Restoring forests in bare areas of high landslide risk and important bird diversity yields benefits for both biodiversity and people. I developed a simple landslide susceptibility model using slope, forest cover, aspect, and stream proximity. Using publicly available bird range maps, refined by elevation, I mapped concentrations of endemic and small-range bird species. I identified 1.54 km2 of potential restoration areas in the Rio Blanco Reserve, and 886 km2 in the Central Andes region. By prioritizing these areas, I facilitate the application of Article 111 which requires local and regional governments to invest in land purchases for the conservation of watersheds.

Chapter 4 dealt with elevational ranges of montane birds and the impact of lowland deforestation on their ranges in the Western Andes of Colombia, an important biodiversity hotspot. Using point counts and mist-nets, I surveyed six altitudinal transects spanning 2200 to 2800m. Three transects were forested from 2200 to 2800m, and three were partially deforested with forest cover only above 2400m. I compared abundance-weighted mean elevation, minimum elevation, and elevational range width. In addition to analyzing the effect of deforestation on 134 species, I tested its impact within trophic guilds and habitat preference groups. Abundance-weighted mean and minimum elevations were not significantly different between forested and partially deforested transects. Range width was marginally different: as expected, ranges were larger in forested transects. Species in different trophic guilds and habitat preference categories showed different trends. These results suggest that deforestation may affect species’ elevational ranges, even within the forest that remains. Climate change will likely exacerbate harmful impacts of deforestation on species’ elevational distributions. Future conservation strategies need to account for this by protecting connected forest tracts across a wide range of elevations.

In Chapter 5, I refine the ranges of 726 species from six biodiversity hotspots by suitable elevation and habitat. This set of 172 bird species for the Atlantic Forest, 138 for Central America, 100 for the Western Andes of Colombia, 57 for Madagascar, 102 for Sumatra, and 157 for Southeast Asia met the criteria for range size, endemism, threat, and forest use. Of these 586 species, the Red List deems 108 to be threatened: 15 critically endangered, 29 endangered, and 64 vulnerable. When ranges are refined by elevational limits and remaining forest cover, 10 of those critically endangered species have ranges < 100km2, but then so do 2 endangered species, seven vulnerable, and eight non-threatened ones. Similarly, 4 critically endangered species, 20 endangered, and 12 vulnerable species have refined ranges < 5000km2, but so do 66 non-threatened species. A striking 89% of these species I have classified in higher threat categories have <50% of their refined ranges inside protected areas. I find that for 43% of the species I assessed, refined range sizes fall within thresholds that typically have higher threat categories than their current assignments. I recommend these species for closer inspection by those who assess risk. These assessments are not only important on a species-by-species basis, but by combining distributions of threatened species, I create maps of conservation priorities. They differ significantly from those created from unrefined ranges.

X-linked inhibitor of apoptosis protein mediates tumor cell resistance to antibody-dependent cellular cytotoxicity.

Inflammatory breast cancer (IBC) is the deadliest, distinct subtype of breast cancer. High expression of epidermal growth factor receptors [EGFR or human epidermal growth factor receptor 2 (HER2)] in IBC tumors has prompted trials of anti-EGFR/HER2 monoclonal antibodies to inhibit oncogenic signaling; however, de novo and acquired therapeutic resistance is common. Another critical function of these antibodies is to mediate antibody-dependent cellular cytotoxicity (ADCC), which enables immune effector cells to engage tumors and deliver granzymes, activating executioner caspases. We hypothesized that high expression of anti-apoptotic molecules in tumors would render them resistant to ADCC. Herein, we demonstrate that the most potent caspase inhibitor, X-linked inhibitor of apoptosis protein (XIAP), overexpressed in IBC, drives resistance to ADCC mediated by cetuximab (anti-EGFR) and trastuzumab (anti-HER2). Overexpression of XIAP in parental IBC cell lines enhances resistance to ADCC; conversely, targeted downregulation of XIAP in ADCC-resistant IBC cells renders them sensitive. As hypothesized, this ADCC resistance is in part a result of the ability of XIAP to inhibit caspase activity; however, we also unexpectedly found that resistance was dependent on XIAP-mediated, caspase-independent suppression of reactive oxygen species (ROS) accumulation, which otherwise occurs during ADCC. Transcriptome analysis supported these observations by revealing modulation of genes involved in immunosuppression and oxidative stress response in XIAP-overexpressing, ADCC-resistant cells. We conclude that XIAP is a critical modulator of ADCC responsiveness, operating through both caspase-dependent and -independent mechanisms. These results suggest that strategies targeting the effects of XIAP on caspase activation and ROS suppression have the potential to enhance the activity of monoclonal antibody-based immunotherapy.