Sascha N. Goonewardena

Folate-targeted nanoparticles show efficacy in the treatment of inflammatory arthritis

OBJECTIVE To investigate the uptake of a poly(amidoamine) dendrimer (generation 5 [G5]) nanoparticle covalently conjugated to polyvalent folic acid (FA) as the targeting ligand into macrophages, and to investigate the activity of an FA- and methotrexate (MTX)-conjugated dendrimer (G5-FA-MTX) as a therapeutic for the inflammatory disease of arthritis. METHODS In vitro studies were performed in macrophage cell lines and in isolated mouse macrophages to check the cellular uptake of fluorescence-tagged G5-FA nanoparticles, using flow cytometry and confocal microscopy. In vivo studies were conducted in a rat model of collagen-induced arthritis to evaluate the therapeutic potential of G5-FA-MTX. RESULTS Folate-targeted dendrimer bound and internalized in a receptor-specific manner into both folate receptor β-expressing macrophage cell lines and primary mouse macrophages. The conjugate G5-FA-MTX acted as a potent antiinflammatory agent and reduced arthritis-induced parameters of inflammation such as ankle swelling, paw volume, cartilage damage, bone resorption, and body weight decrease. CONCLUSION The use of folate-targeted nanoparticles to specifically target MTX into macrophages may provide an effective clinical approach for antiinflammatory therapy in rheumatoid arthritis.

Metabolomics and Atherosclerosis

Metabolites reflect the dynamic processes underlying cellular homeostasis. Recent advances in analytical chemistry and molecular biology have set the stage for metabolite profiling to help us understand complex molecular processes and physiology. Metabolomics is the comparative analysis of metabolite flux and how it relates to biological phenotypes. As an intermediate phenotype, metabolite signatures capture a unique aspect of cellular dynamics that is not typically interrogated, providing a distinct perspective on cellular homeostasis. To date, there have been only a few metabolomics studies investigating cardiovascular diseases. In this review, we explore the principles of metabolomics and how it can provide further insight into the mechanisms of cardiovascular physiology and ultimately lead to improved diagnostic and therapeutic options for patients with cardiovascular disease.

Inflammatory Disequilibrium in Stroke

Over the past several decades, there have been substantial advances in our knowledge of the pathophysiology of stroke. Understanding the benefits of timely reperfusion has led to the development of thrombolytic therapy as the cornerstone of current management of ischemic stroke, but there remains much to be learned about mechanisms of neuronal ischemic and reperfusion injury and associated inflammation. For ischemic stroke, novel therapeutic targets have continued to remain elusive. When considering modern molecular biological techniques, advanced translational stroke models, and clinical studies, a consistent pattern emerges, implicating perturbation of the immune equilibrium by stroke in both central nervous system injury and repair responses. Stroke triggers activation of the neuroimmune axis, comprised of multiple cellular constituents of the immune system resident within the parenchyma of the brain, leptomeninges, and vascular beds, as well as through secretion of biological response modifiers and recruitment of immune effector cells. This neuroimmune activation can directly impact the initiation, propagation, and resolution phases of ischemic brain injury. To leverage a potential opportunity to modulate local and systemic immune responses to favorably affect the stroke disease curve, it is necessary to expand our mechanistic understanding of the neuroimmune axis in ischemic stroke. This review explores the frontiers of current knowledge of innate and adaptive immune responses in the brain and how these responses together shape the course of ischemic stroke.

Flow-dependent expression of ectonucleotide tri(di)phosphohydrolase-1 and suppression of atherosclerosis

The ability of cells to detect and respond to nucleotide signals in the local microenvironment is essential for vascular homeostasis. The enzyme ectonucleotide tri(di)phosphohydrolase-1 (ENTPD1, also known as CD39) on the surface of leukocytes and endothelial cells metabolizes locally released, intravascular ATP and ADP, thereby eliminating these prothrombotic and proinflammatory stimuli. Here, we evaluated the contribution of CD39 to atherogenesis in the apolipoprotein E-deficient (ApoE-deficient) mouse model of atherosclerosis. Compared with control ApoE-deficient animals, plaque burden was markedly increased along with circulating markers of platelet activation in Cd39+/-Apoe-/- mice fed a high-fat diet. Plaque analysis revealed stark regionalization of endothelial CD39 expression and function in Apoe-/- mice, with CD39 prominently expressed in atheroprotective, stable flow regions and diminished in atheroprone areas subject to disturbed flow. In mice, disturbed flow as the result of partial carotid artery ligation rapidly suppressed endothelial CD39 expression. Moreover, unidirectional laminar shear stress induced atheroprotective CD39 expression in human endothelial cells. CD39 induction was dependent upon the vascular transcription factor Krüppel-like factor 2 (KLF2) binding near the transcriptional start site of CD39. Together, these data establish CD39 as a regionalized regulator of atherogenesis that is driven by shear stress.

Design considerations for PAMAM dendrimer therapeutics.

We have previously shown that methotrexate (MTX) conjugated to a cancer-specific poly amido amine (PAMAM) dendrimer has a higher therapeutic index than MTX alone. Unfortunately, these therapeutics have been difficult to advance because of the complicated syntheses and an incomplete understanding of the dendrimer properties. We wished to address these obstacles by using copper-free click chemistry to functionalize the dendrimer scaffolds and to exploring the effects of two dendrimer properties (the targeting ligand and drug linkage) on cytotoxicity. We conjugated either ester or amide-linker modified MTX to dendrimer scaffolds with or without folic acid (FA). Because of multivalency, the FA and MTX functionalized dendrimers had similar capacities to target the folate receptor on cancer cells. Additionally, we found that the ester- and amide-linker modified MTX compounds had similar cytotoxicity but the dendrimer-ester MTX conjugates were much more cytotoxic than the dendrimer-amide MTX conjugates. These results clarify the impact of these properties on therapeutic efficacy and will allow us to design more effective polymer therapeutics.

Monocyte Subsets and Inflammatory Cytokines in Acute Decompensated Heart Failure

BACKGROUND Distinct monocyte subsets predict cardiovascular risk and contribute to heart failure progression in murine models, but they have not been examined in clinical acute decompensated heart failure (ADHF). METHODS AND RESULTS Blood samples were obtained from 11 healthy control subjects (HCs) and at admission and discharge from 19 ADHF patients. Serologic markers of inflammation were assessed at admission and discharge. Monocyte populations were defined with the use of flow cytometry for cell-surface expression of CD14 and CD16: CD14++CD16- (classic), CD14++CD16+ (intermediate), and CD14+CD16++ (nonclassic). In ADHF patients, C-reactive protein (CRP) and interleukin-6 (IL-6) were higher compared with HCs (both P < .001) and decreased from admission to discharge (CRP: 12.1 ± 10.1 to 8.6 ± 8.4 mg/L [P = .005]; IL-6: 19.8 ± 34.5 to 7.1 ± 4.7 pg/mL [P = .08]). In ADHF patients, the admission proportion of CD14++CD16- monocytes was lower (68% vs 85%; P < .001) and that of CD14++CD16+ (15% vs 8%; P = .002) and CD14+CD16++ (17% vs 7%, P = .07) monocytes higher compared with HCs. Additionally, the proportion of CD14++CD16- monocytes increased (68% to 79%, P = .04) and the CD14+CD16++ monocytes decreased (17% to 7%, P = .049) between admission and discharge. CONCLUSIONS Following standard treatment of ADHF, the monocyte profile and circulating inflammatory markers shifts to more closely resemble those of HC, suggesting a resolution of the acute inflammatory state. Functional studies are warranted to understand how specific monocyte subsets and systemic inflammation may contribute to ADHF pathophysiology.

Handcarried Echocardiography to Assess Hemodynamics in Acute Decompensated Heart Failure

Heart failure is a major source of cardiovascular morbidity, including acute decompensations requiring hospitalization. Because most therapeutic interventions in acute heart failure target optimization of cardiac output and volume status, accurate assessment of these parameters at the point of care is critical to guide management. However, physician bedside assessments of left ventricular (LV) function and volume status have limited accuracy. Traditional echocardiographic platforms, while useful for assessing ventricular and valvular function and volume status, have limitations for bedside use or frequent serial evaluation. Handcarried cardiac ultrasound devices, with their substantially lower costs, portability, and ease of use, circumvent many of the limitations of traditional echocardiographic platforms. The diagnostic capabilities of handcarried devices provide the opportunity for ultrasound assessment of LV function and serial bedside evaluation of volume status in patients with acutely decompensated heart failure.

Design and Evaluation of Tumor-Specific Dendrimer Epigenetic Therapeutics

Histone deacetylase inhibitors (HDACi) are promising therapeutics for cancer. HDACi alter the epigenetic state of tumors and provide a unique approach to treat cancer. Although studies with HDACi have shown promise in some cancers, variable efficacy and off-target effects have limited their use. To overcome some of the challenges of traditional HDACi, we sought to use a tumor-specific dendrimer scaffold to deliver HDACi directly to cancer cells. Here we report the design and evaluation of tumor-specific dendrimer–HDACi conjugates. The HDACi was conjugated to the dendrimer using an ester linkage through its hydroxamic acid group, inactivating the HDACi until it is released from the dendrimer. Using a cancer cell model, we demonstrate the functionality of the tumor-specific dendrimer–HDACi conjugates. Furthermore, we demonstrate that unlike traditional HDACi, dendrimer–HDACi conjugates do not affect tumor-associated macrophages, a recently recognized mechanism through which drug resistance emerges. We anticipate that this new class of cell-specific epigenetic therapeutics will have tremendous potential in the treatment of cancer.

Purinergic dysregulation in pulmonary hypertension.

Despite the fact that nucleotides and adenosine help regulate vascular tone through purinergic signaling pathways, little is known regarding their contributions to the pathobiology of pulmonary arterial hypertension, a condition characterized by elevated pulmonary vascular resistance and remodeling. Even less is known about the potential role that alterations in CD39 (ENTPD1), the ectonucleotidase responsible for the conversion of the nucleotides ATP and ADP to AMP, may play in pulmonary arterial hypertension. In this study we identified decreased CD39 expression on the pulmonary endothelium of patients with idiopathic pulmonary arterial hypertension. We next determined the effects of CD39 gene deletion in mice exposed to normoxia or normobaric hypoxia (10% oxygen). Compared with controls, hypoxic CD39(-/-) mice were found to have a markedly elevated ATP-to-adenosine ratio, higher pulmonary arterial pressures, more right ventricular hypertrophy, more arterial medial hypertrophy, and a pro-thrombotic phenotype. In addition, hypoxic CD39(-/-) mice exhibited a marked increase in lung P2X1 receptors. Systemic reconstitution of ATPase and ADPase enzymatic activities through continuous administration of apyrase decreased pulmonary arterial pressures in hypoxic CD39(-/-) mice to levels found in hypoxic CD39(+/+) controls. Treatment with NF279, a potent and selective P2X1 receptor antagonist, lowered pulmonary arterial pressures even further. Our study is the first to implicate decreased CD39 and resultant alterations in circulating purinergic signaling ligands and cognate receptors in the pathobiology of pulmonary arterial hypertension. Reconstitution and receptor blocking experiments suggest that phosphohydrolysis of purinergic nucleotide tri- and diphosphates, or blocking of the P2X1 receptor could serve as treatment for pulmonary arterial hypertension.

Therapeutic Impact of Nanoparticle Therapy Targeting Tumor-Associated Macrophages

Antiangiogenic therapies, despite initial encouragement, have demonstrated a limited benefit in ovarian cancer. Laboratory studies suggest antiangiogenic therapy–induced hypoxia can induce tumor “stemness” as resistance to antiangiogenic therapy develops and limits the therapeutic benefit. Resistance to antiangiogenic therapy and an induction of tumor stemness may be mediated by proangiogenic tumor-associated macrophages (TAM). As such, TAMs have been proposed as a therapeutic target. We demonstrate here that ovarian TAMs express high levels of the folate receptor-2 (FOLR2) and can be selectively targeted using G5-dendrimer nanoparticles using methotrexate as both a ligand and a toxin. G5-methotrexate (G5-MTX) nanoparticles deplete TAMs in both solid tumor and ascites models of ovarian cancer. As a therapeutic agent, these nanoparticles are more effective than cisplatin. Importantly, these nanoparticles could (i) overcome resistance to antiangiogenic therapy, (ii) prevent antiangiogenic therapy–induced increases in cancer stem–like cells in both murine and human tumor cell models, (iii) prevent antiangiogenic therapy–induced increases in VEGF-C, and (iv) prevent antiangiogenic therapy–induced BRCA1 gene expression. Combined, this work strongly supports the development of TAM-targeted nanoparticle therapy. Mol Cancer Ther; 17(1); 96–106. ©2017 AACR.