Targeting CD34(+) cells of the inflamed synovial endothelium by guided nanoparticles for the treatment of rheumatoid arthritis

Despite the advances in the treatment of rheumatoid arthritis (RA) achieved in the last few years, several patients are diagnosed late, do not respond to or have to stop therapy because of inefficacy and/or toxicity, leaving still a huge unmet need. Tissue-specific strategies have the potential to address some of these issues. The aim of the study is the development of a safe nanotechnology approach for tissue-specific delivery of drugs and diagnostic probes. CD34 + endothelial precursors were addressed in inflamed synovium using targeted biodegradable nanoparticles (tBNPs). These nanostructures were made of poly-lactic acid, poly-caprolactone, and PEG and then coated with a synovial homing peptide. Immunofluorescence analysis clearly demonstrated their capacity to selectively address CD34 + endothelial cells in synovial tissue obtained from human, mouse, and rat. Biodistribution studies in two different animal models of rheumatoid arthritis (antigen-induced arthritis/AIA and collagen-induced arthritis/CIA) confirmed the selective accumulation in inflamed joints but also evidenced the capacity of tBNP to detect early phases of the disease and the preferential liver elimination. The therapeutic effect of methotrexate (MTX)-loaded tBNPs were studied in comparison with conventional MTX doses. MTX-loaded tBNPs prevented and treated CIA and AIA at a lower dose and reduced administration frequency than MTX. Moreover, MTX-loaded tBNP showed a novel mechanism of action, in which the particles target and kill CD34 + endothelial progenitors, preventing neo-angiogenesis and, consequently, synovial inflammation. tBNPs represent a stable and safe platform to develop highly-sensitive imaging and therapeutic approaches in RA targeting specifically synovial neo-angiogenesis to reduce local inflammation

Expression of genes involved in lipid biosynthesis pathway in white adipose tissue during cancer cachexia.

<p>mRNA analysis of <i>Pparγ</i>, <i>C/ebpα</i>, <i>Lpl</i>, <i>Fas</i>, <i>Scd1</i>, <i>Dgat2</i>, isolated from WAT of control (white circle), and C26-bearing mice (black square); Values are mean ± s.e.m. presented as percentages relative to the controls for 4–5 animals per group (*P<0.05; **P<0.01; ***P<0.001 C26 vs control). 6 am values are duplicated in each graph to illustrate a complete 24-h cycle.</p

Involvement of AMPK and mTOR/4EBP1 signaling pathways in cancer cachexia.

<p>(A) mRNA analysis of <i>Ampkα1, Ampkα2</i> and <i>Acc</i> from WAT of control (white circle), and C26-bearing mice (black square); (B) Western blot analysis of phospho-AMPK (Thr192) and total AMPK at 2 pm and 2 am; Immunoblot analysis of Phospho-ACC and total ACC for 2 pm WAT samples. (C) mRNA analysis of <i>mTor</i> and <i>4Ebp1;</i> from WAT of control (white circle), and C26-bearing mice (black square); (D) Western blot analysis of phospho-mTOR (Ser2448) total mTOR, phospho-4EBP1 (Ser65), phospho-4EBP1 (Thr37/46) and total 4EBP1 at 2 pm and 2 am. Values are mean ± s.e.m. presented as percentages relative to the controls for 4–5 animals per group (*P<0.05; **P<0.01; C26 vs control). 6 am values are duplicated in each graph to illustrate a complete 24-h cycle.</p

Lipolytic pathway in WAT of cachectic animals.

<p>(A) mRNA analysis of <i>Hsl</i> gene expression and Western blot analysis of phospho-PKA (Thr197), total PKA, phospho-HSL and total HSL proteins at 2 am and 2 pm; (B) mRNA analysis of <i>Atgl</i> and <i>Perilipin</i> gene expression from WAT of control (white circle), and C26 tumour-bearing mice (black square); Values are mean ± s.e.m. presented as percentages relative to the controls for 4–5 animals per group (*P<0.05; **P<0.01; ***P<0.001, C26 vs control). 6 am values are duplicated in each graph to illustrate a complete 24-h cycle; (C) Western blot analysis of ATGL and PERILIPIN proteins at 2 pm and 2 am.</p

Expression of core clock genes in white adipose tissue during cancer cachexia.

<p>mRNA analysis of <i>Rev-erbα</i>, <i>Bmal</i>, <i>Per2</i> and <i>Cry1</i> isolated from WAT of control (white circle), and C26-bearing mice (black square); Values are mean ± s.e.m. presented as percentages relative to the controls for 4–5 animals per group (*P<0.05; **P<0.01; ***P<0.001 C26 vs control). 6 am values are duplicated in each graph to illustrate a complete 24-h cycle.</p

Depletion of White Adipose Tissue in Cancer Cachexia Syndrome Is Associated with Inflammatory Signaling and Disrupted Circadian Regulation

<div><p>Involuntary weight loss in patients with cancer is the hallmark of cancer cachexia. The etiology of cachexia is multifactorial involving loss of skeletal muscle and adipose tissue associated with high systemic levels of acute phase proteins and inflammatory cytokines. While muscle wasting overtly impacts on cancer patient quality of life, loss of lipid depots represents a sustained energy imbalance. In this study fat depletion was examined in Colon-26 model of cancer cachexia, which is a widely used rodent model of this syndrome. We investigated diurnal expression of circadian rhythm regulators as well as key mediators of energy metabolism and cytokine signaling. Mice bearing the C26 tumour exhibited reduced adipose mass, elevated adipose tissue lipolysis and a 5-fold increase in plasma levels of free fatty acids. These changes were associated with activated IL-6 signaling in WAT through a 3-fold increase in phosphorylated STAT3 and high SOCS3 gene expression levels. In addition perturbations in circadian regulation of lipid metabolism were also observed. Lipid catabolism did not appear to be influenced by the classical PKA pathway activating the lipase HSL. ATGL protein levels were elevated 2-fold in cachectic mice while 4-fold increase phosphorylated ACC and a 2-fold decrease in phosphorylated 4EBP1 was observed indicating that lipid metabolism is modulated by the ATGL & AMPK/mTOR pathways. This study provides evidence for activation of cytokine signaling and concomitant alterations in circadian rhythm and regulators of lipid metabolism in WAT of cachectic animals.</p></div

Activation of IL-6 signaling pathway in C26-bearing mice.

<p>Diurnal mRNA expression of SOCS3 mRNA in white adipose tissue from control mice (white circle) and cachectic mice (black square) (^*P<0.05, ^**P<0.01, ^***P<0.001 vs control). 6 am values are duplicated in the graph to illustrate diurnal rhythmicity. mRNA expression values are mean ± s.e.m. relative to controls for 4–6 animals per group. Western blot and densitometric analysis of STAT3 protein and phospho-STAT3 (Ser727), in white adipose tissue from cachectic and control animals.</p

Supplementary Figure 4 from Microtubule-Targeting Combined with HDAC Inhibition Is a Novel Therapeutic Strategy for Diffuse Intrinsic Pontine Gliomas

SAHA significantly affects alpha acetyl tubulin and HDAC6 levels in DIPG.</p

Diurnal expression of genes in lipid utilization pathways in cachectic mice.

<p>(A) mRNA analysis of <i>Pparα, Pgc1α, Pparδ</i>, <i>Nrf1, Tfam, Cpt1α</i> and <i>Pbe</i> from WAT of control (white circle) and C26 tumour -bearing mice (black square); Values are mean ± s.e.m. presented as percentages relative to the controls for 4–5 animals per group (*P<0.05; **P<0.01; C26 vs control). 6 am values are duplicated in each graph to illustrate a complete 24-h cycle; (B) Western blot analysis of PBE protein at 2 pm and 2 am time points.</p