Prolymphangiogenic Effects of 9-cis Retinoic Acid Are Enhanced at Sites of Lymphatic Injury and Dependent on Treatment Duration in Experimental Postsurgical Lymphedema.

Background: Patients undergoing surgical treatment for solid tumors are at risk for development of secondary lymphedema due to intraoperative lymphatic vessel injury. The damaged lymphatic vessels fail to adequately regenerate and lymphatic obstruction leads to fluid and protein accumulation in the interstitial space and chronic lymphedema develops as a result. There are currently no effective pharmacological agents that reduce the risk of developing lymphedema or treat pre-existing lymphedema, and management is largely palliative. The present study investigated the efficacy of various 9-cis retinoic acid (9-cis RA) dosing strategies in reducing postsurgical lymphedema by utilizing a well-established mouse tail lymphedema model. Methods and Results: Short-duration treatment with 9-cis RA did not demonstrate a significant reduction in postoperative tail volume, nor an improvement in lymphatic clearance. However, long-term treatment with 9-cis RA resulted in decreased overall tail volume, dermal thickness, and epidermal thickness, with an associated increase in functional lymphatic clearance and lymphatic vessel density, assessed by LYVE-1 immunostaining, compared with control. These effects were seen at the site of lymphatic injury, with no significant changes observed in uninjured sites such as ear skin and the diaphragm. Conclusions: Given the reported results indicating that 9-cis RA is a potent promoter of lymphangiogenesis and improved lymphatic clearance at sites of lymphatic injury, investigation of postoperative 9-cis RA administration to patients at high risk of developing lymphedema may demonstrate positive efficacy and reduced rates of postsurgical lymphedema.

The MS Remyelinating Drug Bexarotene (an RXR Agonist) Promotes Induction of Human Tregs and Suppresses Th17 Differentiation In Vitro.

The retinoid X receptor agonist bexarotene promotes remyelination in patients with multiple sclerosis. Murine studies have also demonstrated that RXR agonists have anti-inflammatory effects by enhancing the ability of all-trans-retinoic acid (atRA) to promote T-regulatory cell (Treg) induction and reduce Th17 differentiation in vitro. By stimulating human naïve CD4 T-cells in the presence of Treg or Th17 skewing cytokines, we show that bexarotene also tips the human Treg/Th17 axis in favor of Treg induction, but unlike murine cells this occurs independently of atRA and retinoic acid receptor signaling. Tregs induced in the presence of bexarotene express canonical markers of T-regulation and are functionally suppressive in vitro. Circulating Treg numbers did not increase in the blood of trial patients receiving bexarotene; we believe this is because Treg induction is likely to occur within tissues. These findings lend support to developing RXR agonists as treatments of autoimmune diseases, in particular multiple sclerosis.

Dual effects of 9-cis retinoic acid on ACTH-dependent hyperplastic adrenal tissues.

Retinoids play a pivotal role in adrenal development and differentiation. Recent clinical trials revealed therapeutic potential of both all-trans and 9-cis retinoic acid in patients with cortisol excess due to a pituitary ACTH-secreting adenoma and indicated that retinoids might act also on the adrenal. Aim of the present study was to evaluate the effect of 9-cis retinoic acid on adrenals from patients with ACTH-dependent Cushing's syndrome. Adrenal specimens from six patients with Cushing's disease were incubated with 10 nM-1 µM 9-cis retinoic acid with and without 10 nM ACTH. Cortisol secretion was measured by immunoassay and expression of genes involved in steroidogenesis as well as retinoic acid action were evaluated by real-time RT-PCR. Incubation with 10-100 nM 9-cis retinoic acid increased spontaneous cortisol secretion and expression of STAR and CYP17A. On the other hand, in wells treated with ACTH, 9-cis retinoic acid markedly diminished ACTH receptor upregulation and no stimulatory effect on cortisol secretion or steroidogenic enzyme synthesis was observed. ACTH itself increased ligand-induced retinoic acid receptor expression, possibly enhancing sensitivity to retinoic acid. Our findings indicate that the effect of 9-cis retinoic acid in presence of ACTH is distinct from unchallenged wells and support the hypothesis of a direct adrenal action in patients with Cushing's disease.

9-cis Retinoic acid and 1.25-dihydroxyvitamin D3 drive differentiation into IgA+ secreting plasmablasts in human naïve B cells.

Calcitriol and 9-cis retinoic acid (9cRA) play a fundamental role in shaping the adaptive immune response by altering the Ig profile and the differentiation of B cells, controlled by their corresponding nuclear receptors, VDR and RAR. Herein, after the establishment of a plasmablast differentiation culture, we investigated how both ligands modulate human naïve B cell differentiation and to which extent VDR/RXR and RAR/RXR signaling interferes. Calcitriol and 9cRA mediated activation of purified naïve B cells resulted in a strong differentiation of CD27+ CD38+ plasmablasts and antibody secretion. The significant IgA response was preceded by a strong induction of α-germline transcription (GLT). Induction of αGLT and consecutively IgA secretion driven by calcitriol is a novel observation and we show by magnetic chromatin IP that this was mediated by recruitment of the VDR to the TGF-ß promoter thus inducing TGF-ß expression. Finally, as revealed by transcriptomic profiling calcitriol and 9cRA modulate several signals required for differentiation and isotype switching in a noncompeting but rather additive manner. Calcitriol and 9cRA participate in the control of the IgA response in human activated naïve B cells. The balance between both ligands may be an important factor in channeling humoral immune responses toward a protective direction.

Intranasal delivery of 9-cis retinoic acid reduces beta-amyloid deposition via inhibiting astrocyte-mediated inflammation.

Alzheimer's disease (AD) is associated with the accumulation and deposition of a beta-amyloid (Αß) peptide in the brain, resulting in increased neuroinflammation and synaptic dysfunction. Intranasal delivery of targeted drugs to the brain represents a noninvasive pathway that bypasses the blood-brain barrier and minimizes systemic exposure. The aim of this study was to evaluate the therapeutic effect of intranasally delivered 9-cis retinoic acid (RA) on the neuropathology of an AD mouse model. Herein, we observed dramatically decreased Αß deposition in the brains of amyloid precursor protein (APP) and presenilin 1 (PS1) double-transgenic mice (APP/PS1) treated intranasally with 9-cis RA for 4 weeks compared to that in the brains of vehicle-treated mice. Importantly, intranasal delivery of 9-cis RA suppressed Αß-associated astrocyte activation and neuroinflammation and ultimately restored synaptic deficits in APP/PS1 transgenic mice. These results support the critical roles of Αß-associated neuroinflammation responses to synaptic deficits, particularly during the deposition of Αß. Our findings provide strong evidence that intranasally delivered 9-cis RA attenuates neuronal dysfunction in an AD mouse model and is a promising therapeutic strategy for the prevention and treatment of AD.

VHL enhances 9-cis-retinoic acid treatment by down-regulating retinoid X receptor α in renal cell carcinomas.

Renal cell carcinoma (RCC) is the most common malignant kidney tumors in adults. Von Hippel-Lindau (VHL) gene is deficient in >50% of RCC cases, but the role of VHL as a potential therapeutic target in RCC has not been well established. In the present study, 9-cis-Retinoic acid, which is a potent natural agonist of retinoid X receptors (RXRs), was found to decrease the viability of VHL-proficient RCC cells, but had little effect on VHL-deficient RCC cells. In addition, it was demonstrated that VHL transcriptionally regulated RXRα in a hypoxia-inducible factor-α independent manner. Moreover, a negative correlation was observed between the expressions of VHL and RXRα in RCC tissues. Collectively, these data indicate that VHL-proficient RCC patients may be more sensitive to treatment with 9-cis-retinoic acid, which acts by regulating RXRα expression, compared with VHL-deficient RCC patients. The findings of the present study demonstrate a novel function of VHL and highlight the potential of VHL expression as a therapeutic modality for the optimized treatment of RCC patients.

Chemical constituents from the deep sea-derived Streptomyces xiamenensis MCCC 1A01570 and their effects on RXRα transcriptional regulation.

From the deep sea-derived Streptomyces xiamenensis MCCC 1A01570, eight cyclic dipeptides (1-8) and five phenolics (9-13) were obtained. Cyclo-(I-Pro-D-Leu) (4) could moderately promote the gene transcriptional function of nuclear receptor RXRα, while 2, 3, and 13 showed weak reduction in RXRα gene transcriptional activities induced by 9-cis-retinoid acid (RA). These compounds might have beneficial effects against intractable diseases with relation to RXRα, such as cancer and metabolic diseases, due to their potential activities on regulating the transcriptional activation function of RXRα. In addition, 1-6, 8, 10, and 12 (20 µM) showed mild in vitro cytotoxicity against three cancer cell lines of ECA-109, Hela-S3 and PANC-1 with the inhibition rates arranging from 50% to 65%.

Comparison of the effect of isotretionin and alitretionin on endometriotic implants and serum vascular endothelial growth factor level: an experimental study.

OBJECTIVE: To compare the effects of alitretionin and isotretionin on endometrial peritoneal implants and serum vascular endothelial growth factor (VEGF) levels. STUDY DESIGN: Forty-eight female Sprague Dawley rats were used. Initially surgical rat endometriosis model was done. The endometrial implant volume was measured and rats were randomly divided into four groups. Group 1: Control group (rats did not get any drug but having endometriotic implants), group 2: rats receiving po isotretionin 10 mg/kg per day for 10 d, group 3: rats receiving po isotretionin 20 mg/kg per day for 10 d and group 4: rats receiving po alitretionin 80 mg/kg per day for 10 d. After 1-week medication, rats were sacrificed and size, histopathology of endometriotic implant and levels of VEGF were evaluated. RESULTS: Volumes of peritoneal endometrial implants were significantly decreased in Group 2 and Group 3 compared with initial values. However, there were no significant changes in histopathological scores and serum VEGF levels in all groups. CONCLUSIONS: This study finding may suggest the possible medical treatment modality of isotretionin on endometriosis. However, alitretionin (potent retinoid) does not have potent regressive effect on endometriotic implants as in isotretionin.

Small Peptide Modulation of Fibroblast Growth Factor Receptor 3-Dependent Postnatal Lymphangiogenesis.

BACKGROUND: The fibroblast growth factor receptor (FGFR) family includes transmembrane receptors involved in a wide range of developmental and postdevelopmental biologic processes as well as a wide range of human diseases. In particular, FGFR3 has been implicated in the mechanism by which 9-cis retinoic acid (9-cisRA) induces lymphangiogenesis and improves lymphedema. The purpose of this study was to validate the efficacy of a novel small peptide FGFR3 inhibitor, peptide P3 (VSPPLTLGQLLS), and to elucidate the role of FGFR3 in 9-cisRA-induced lymphangiogenesis using this peptide. METHODS AND RESULTS: Peptide P3 effectively inhibited FGFR3 phosphorylation. In vitro, peptide P3-mediated FGFR3 inhibition did not decrease lymphatic endothelial cell (LEC) proliferation, migration, or tubule formation. However, peptide P3-mediated FGFR3 inhibition did block 9-cisRA-stimulated LEC proliferation, migration, and tubule formation. In vivo, peptide P3-mediated FGFR3 inhibition was sufficient to inhibit 9-cisRA-induced tracheal lymphangiogenesis. CONCLUSION: FGFR3 does not appear to be essential to nonpromoted LEC proliferation, migration, and tubule formation. However, FGFR3 may play a key role in LEC proliferation, migration, tubule formation, and postnatal in vivo lymphangiogenesis when pharmacologically induced by 9-cisRA. P3 may have the potential to be used as a precise regulatory control element for 9-cisRA-mediated lymphangiogenesis.

A Novel Dendritic Cell-Based Vaccination Protocol to Stimulate Immunosurveillance of Aggressive Cancers.

A major challenge in the development of a successful tumor vaccination is to break immune tolerance and to sensitize efficiently the immune system toward relevant tumor antigens, thus enabling T-cell-mediated antitumor responses in vivo. Dendritic cell (DC)-based immunotherapy shows the advantage to induce an adaptive immune response against the tumor, with the potential to generate a long-lasting immunological memory able to prevent further relapses and hopefully metastasis. Recently different preclinical studies highlighted the golden opportunity to exploit the features of immunogenic cell death (ICD) to generate ex vivo a highly immunogenic tumor cell lysate as potent antigen formulation for improved DC-based vaccine against aggressive cancers. This chapter focuses on the methods to obtain tumor lysates from cells undergoing ICD to be used for DC pulsing and to test the functionality of the generated DCs for antitumor vaccine development.

Effects of 9-cis-retinoic acid on the proliferation and apoptosis of cutaneous T-cell lymphoma cells.

The vitamin A derivative 9-cis-retinoic acid (9-cis-RA) has been used for the treatment and prevention of cutaneous T-cell lymphoma (CTCL). However, the precise mechanism by which 9-cis-RA treatment ameliorates CTCL remains elusive. Our research shows that 9-cis-RA inhibits proliferation and induces apoptosis in CTCL cells in a dose-dependent and time-dependent manner. 9-Cis-RA also induced G0/G1 cell cycle arrest by downregulation of cyclin D1. We confirmed that 9-cis-RA significantly decreased phosphorylation of JAK1, STAT3, and STAT5 and downregulated Bcl-xL and cyclin D1, indicating that 9-cis-RA inhibited the activation of JAK/STAT signaling. Meanwhile, 9-cis-RA also activated classical RA-mediated transcription by retinoic acid receptors (RAR) and/or retinoid X receptors (RXR) in a CTCL cell line. Thus, 9-cis-RA may be effective for chemotherapy and may prevent human CTCL by inhibiting proliferation and inducing apoptosis by inhibition of the JAK/STAT pathway and activation of the RAR/RXR pathway.

9-cis retinoic acid modulates the type I allergic immune response.

BACKGROUND: Vitamin A is a potent regulator of adaptive immunity. The effect of the endogenous metabolite 9-cis retinoic acid (9cRA) on allergic sensitization is unknown. OBJECTIVE: We sought to investigate whether and to what extent 9cRA modulates the humoral immune response. METHODS: BALB/c mice were sensitized and challenged with ovalbumin (OVA). 9cRA was applied repeatedly together with the antigen. Immunoglobulin production and cellular analysis were performed by using ELISA, ELISpot, and flow cytometry. Human CD19+ B cells were activated in vitro in the presence or absence of 9cRA and activation markers, and proliferation and secreted immunoglobulin levels were analyzed by using flow cytometry and ELISA. RESULTS: 9cRA applied together with repeated OVA challenge transiently increased specific serum IgA, IgE, and IgG1 serum levels (2.0- and 8.9-fold). After OVA recall, specific IgE concentrations were reduced by a mean of 57% after adding 9cRA, whereas IgA was strongly induced (20-fold), and IgG1 levels remained unchanged. Correspondingly, less specific IgE- and more IgA-secreting cells resided in the spleen in the 9cRA groups. Additionally, 9cRA promoted the migration of specific B cells to the mesenteric but not draining lymph nodes. In purified stimulated human B cells, 9cRA markedly reduced IgE production and enhanced IgA production. B-cell activation was modulated by 9cRA, reducing the expression of CD86 and promoting IL-10. CONCLUSIONS: Our data indicate that 9cRA modulates the allergic immune response by reducing the IgE response but promoting the IgA response. Thus 9cRA can modulate the allergic immune response toward a non-IgE condition.

9-cis retinoic acid induces neurorepair in stroke brain.

The purpose of this study was to examine the neurorestorative effect of delayed 9 cis retinoic acid (9cRA) treatment for stroke. Adult male rats received a 90-min right distal middle cerebral artery occlusion (dMCAo). Animals were separated into two groups with similar infarction sizes, based on magnetic resonance imaging on day 2 after dMCAo. 9cRA or vehicle was given via an intranasal route daily starting from day 3. Stroke rats receiving 9cRA post-treatment showed an increase in brain 9cRA levels and greater recovery in motor function. 9cRA enhanced the proliferation of bromodeoxyuridine (+) cells in the subventricular zone (SVZ) and lesioned cortex in the stroke brain. Using subventricular neurosphere and matrigel cultures, we demonstrated that proliferation and migration of SVZ neuroprogenitor cells were enhanced by 9cRA. Our data support a delayed and non-invasive drug therapy for stroke. Intranasal 9cRA can facilitate the functional recovery and endogenous repair in the ischemic brain.