Study of the role of skin lymphatics in electrolyte and blood pressure regulation

Cardiovascular diseases are the major cause of death worldwide and represent a dramatic socio-economic challenge. Hypertension accounts for 18% of cardiovascular disease deaths in the Western countries, and is a major risk factor for stroke, coronary heart disease and heart failure. Excessive dietary salt intake is known to be a risk factor for developing hypertension, but the pathophysiology of salt sensitive hypertension is poorly understood. The kidneys are the main regulators of Na+ and water in the body. Salt sensitive hypertension has traditionally been explained by an impaired capacity of the kidneys to excrete Na+, resulting in water retention and thereby a progressive alteration in the filling of the vasculature, resulting in increased blood pressure. Recent studies have suggested that Na+ can be retained or removed from the body without commensurate water, and that the skin may function as sodium reservoir. It has been shown that the Na+ accumulation is controlled by immune cells and involves modification of the extracellular matrix and lymphangiogenesis in the skin. In this thesis we therefore addressed three major questions to clarify aspects of the new hypothesis proposing the skin as a contributor to Na+ and blood pressure homeostasis; 1) What are the microcirculatory effects of increased lymphatic vasculature in the skin, 2) are new lymph vessels induced by Na+ retention functional, and 3) does lymphatic vasculature in the skin affect Na+ accumulation and blood pressure homeostasis. To study the microcirculatory effects of a chronically expanded lymphatic vasculature in the skin we used K14-VEGF-C mice overexpressing vascular endothelial growth factor-C (VEGF-C), resulting in an expanded lymphatic network in skin. Acute and chronic inflammation resulted in increased interstitial fluid pressure and reduced lymph flow, but to the same extent in transgenic mice and WT controls. However, after local overhydration in the skin we observed increased lymph flow and fluid transport in the transgenic mice. Despite increased production of the immune cell chemoattractant CCL21 in K14-VEGF-C mice, local inflammation did not result in an increased number of migrating immune cells from the skin to the draining lymph node. We concluded that lymphangiogenesis might enhance clearance of fluid in situations with increased fluid filtration. Sodium accumulation in the skin is suggested to be regulated by macrophages that secrete VEGF-C in response to a hyperosmotic microenvironment thereby stimulating lymphangiogenesis. An important question is whether these newly formed vessels are functional. After salt loading in rats we measured lymph flow in skin and muscle with optical imaging and a newly developed PET-CT method. Increased lymph flow was observed in skin as well as muscle. A reduction of lymph flow was observed after macrophage depletion in the skin. Our findings suggest that newly formed lymphatic vessels are functional, and that macrophages may be involved in the regulation of lymph flow and thereby clearance of Na+ from tissues. Previous studies have shown that mice lacking lymphatics in the skin develop higher blood pressure after salt loading. To address the question whether lymphatic vasculature in skin is important for Na+ accumulation and blood pressure homeostasis, we used genetically engineered mice with either increased or reduced lymphatic vasculature in the skin. Blood pressure was measured with telemetric recording before salt loading and at the termination of the experiment. Tissue samples from skin and muscle were harvested for analysis of Na+ and K+ concentration. We found no differences in Na+ accumulation or blood pressure response between genetically engineered mice and normal controls. Our results suggest that lymphatic vasculature in skin does not have an important role in electrolyte and blood pressure homeostasis in mice

The Protein Kinase C Agonist PEP005 (Ingenol 3-Angelate) in the Treatment of Human Cancer: A Balance between Efficacy and Toxicity

The diterpene ester ingenol-3-angelate (referred to as PEP005) is derived from the plant Euphorbia peplus. Crude euphorbia extract causes local toxicity and transient inflammation when applied topically and has been used in the treatment of warts, skin keratoses and skin cancer. PEP005 is a broad range activator of the classical (α, β, γ) and novel (δ, ε, η, θ) protein kinase C isoenzymes. Direct pro-apoptotic effects of this drug have been demonstrated in several malignant cells, including melanoma cell lines and primary human acute myelogenous leukemia cells. At micromolar concentrations required to kill melanoma cells this agent causes PKC-independent secondary necrosis. In contrast, the killing of leukemic cells occurs in the nanomolar range, requires activation of protein kinase C δ (PKCδ) and is specifically associated with translocation of PKCδ from the cytoplasm to the nuclear membrane. However, in addition to this pro-apoptotic effect the agent seems to have immunostimulatory effects, including: (i) increased chemokine release by malignant cells; (ii) a general increase in proliferation and cytokine release by activated T cells, including T cells derived from patients with chemotherapy-induced lymphopenia; (iii) local infiltration of neutrophils after topical application with increased antibody-dependent cytotoxicity; and (iv) development of specific anti-cancer immune responses by CD8+ T cells in animal models. Published studies mainly describe effects from in vitro investigations or after topical application of the agent, and careful evaluation of the toxicity after systemic administration is required before the possible use of this agent in the treatment of malignancies other than skin cancers

Study of the role of skin lymphatics in electrolyte and blood pressure regulation

Cardiovascular diseases are the major cause of death worldwide and represent a dramatic socio-economic challenge. Hypertension accounts for 18% of cardiovascular disease deaths in the Western countries, and is a major risk factor for stroke, coronary heart disease and heart failure. Excessive dietary salt intake is known to be a risk factor for developing hypertension, but the pathophysiology of salt sensitive hypertension is poorly understood. The kidneys are the main regulators of Na+ and water in the body. Salt sensitive hypertension has traditionally been explained by an impaired capacity of the kidneys to excrete Na+, resulting in water retention and thereby a progressive alteration in the filling of the vasculature, resulting in increased blood pressure. Recent studies have suggested that Na+ can be retained or removed from the body without commensurate water, and that the skin may function as sodium reservoir. It has been shown that the Na+ accumulation is controlled by immune cells and involves modification of the extracellular matrix and lymphangiogenesis in the skin. In this thesis we therefore addressed three major questions to clarify aspects of the new hypothesis proposing the skin as a contributor to Na+ and blood pressure homeostasis; 1) What are the microcirculatory effects of increased lymphatic vasculature in the skin, 2) are new lymph vessels induced by Na+ retention functional, and 3) does lymphatic vasculature in the skin affect Na+ accumulation and blood pressure homeostasis. To study the microcirculatory effects of a chronically expanded lymphatic vasculature in the skin we used K14-VEGF-C mice overexpressing vascular endothelial growth factor-C (VEGF-C), resulting in an expanded lymphatic network in skin. Acute and chronic inflammation resulted in increased interstitial fluid pressure and reduced lymph flow, but to the same extent in transgenic mice and WT controls. However, after local overhydration in the skin we observed increased lymph flow and fluid transport in the transgenic mice. Despite increased production of the immune cell chemoattractant CCL21 in K14-VEGF-C mice, local inflammation did not result in an increased number of migrating immune cells from the skin to the draining lymph node. We concluded that lymphangiogenesis might enhance clearance of fluid in situations with increased fluid filtration. Sodium accumulation in the skin is suggested to be regulated by macrophages that secrete VEGF-C in response to a hyperosmotic microenvironment thereby stimulating lymphangiogenesis. An important question is whether these newly formed vessels are functional. After salt loading in rats we measured lymph flow in skin and muscle with optical imaging and a newly developed PET-CT method. Increased lymph flow was observed in skin as well as muscle. A reduction of lymph flow was observed after macrophage depletion in the skin. Our findings suggest that newly formed lymphatic vessels are functional, and that macrophages may be involved in the regulation of lymph flow and thereby clearance of Na+ from tissues. Previous studies have shown that mice lacking lymphatics in the skin develop higher blood pressure after salt loading. To address the question whether lymphatic vasculature in skin is important for Na+ accumulation and blood pressure homeostasis, we used genetically engineered mice with either increased or reduced lymphatic vasculature in the skin. Blood pressure was measured with telemetric recording before salt loading and at the termination of the experiment. Tissue samples from skin and muscle were harvested for analysis of Na+ and K+ concentration. We found no differences in Na+ accumulation or blood pressure response between genetically engineered mice and normal controls. Our results suggest that lymphatic vasculature in skin does not have an important role in electrolyte and blood pressure homeostasis in mice

Distinct single cell signal transduction signatures in leukocyte subsets stimulated with khat extract, amphetamine-like cathinone, cathine or norephedrine

Background: Amphetamine and amphetamine derivatives are suggested to induce an immunosuppressive effect. However, knowledge of how amphetamines modulate intracellular signaling pathways in cells of the immune system is limited. We have studied phosphorylation of signal transduction proteins (Akt, CREB, ERK1/2, NF-κB, c-Cbl, STAT1/3/5/6) and stress sensors (p38 MAPK, p53) in human leukocyte subsets following in vitro treatment with the natural amphetamine cathinone, the cathinone derivatives cathine and norephedrine, in comparison with a defined extract of the psychostimulating herb khat (Catha edulis Forsk.). Intracellular protein modifications in single cells were studied using immunostaining and flow cytometry, cell viability was determined by Annexin V-FITC/Propidium Iodide staining, and T-lymphocyte proliferation was measured by 3H-thymidine incorporation. Results: Cathinone, cathine and norephedrine generally reduced post-translational modifications of intracellular signal transducers in T-lymphocytes, B-lymphocytes, natural killer cells and monocytes, most prominently affecting c-Cbl (pTyr700), ERK1/2 (p-Thr202/p-Tyr204), p38 MAPK (p-Thr180/p-Tyr182) and p53 (both total p53 protein and p- Ser15). In contrast, the botanical khat-extract induced protein phosphorylation of STAT1 (p-Tyr701), STAT6 (p- Tyr641), c-Cbl (pTyr700), ERK1/2 (p-Thr202/p-Tyr204), NF-κB (p-Ser529), Akt (p-Ser473), p38 MAPK (p-Thr180/p- Tyr182), p53 (Ser15) as well as total p53 protein. Cathinone, cathine and norephedrine resulted in unique signaling profiles, with B-lymphocytes and natural killer cells more responsive compared to T-lymphocytes and monocytes. Treatment with norephedrine resulted in significantly increased T-lymphocyte proliferation, whereas khat-extract reduced proliferation and induced cell death. Conclusions: Single-cell signal transduction analyses of leukocytes distinctively discriminated between stimulation with cathinone and the structurally similar derivatives cathine and norephedrine. Cathinone, cathine and norephedrine reduced phosphorylation of c-Cbl, ERK1/2, p38 MAPK and p53(Ser15), and norephedrine induced Tlymphocyte proliferation. Khat-extract induced protein phosphorylation of signal transducers, p38 MAPK and p53, followed by reduced cell proliferation and cell death. This study suggests that protein modification-specific singlecell analysis of immune cells could unravel pharmacologic effects of amphetamines and amphetamine-like agents, and further could represent a valuable tool in elucidation of mechanism(s) of action of complex botanical extracts

Molecular mechanisms of nutlin-3 involve acetylation of p53, histones and heat shock proteins in acute myeloid leukemia

Background The small-molecule MDM2 antagonist nutlin-3 has proved to be an effective p53 activating therapeutic compound in several preclinical cancer models, including acute myeloid leukemia (AML). We and others have previously reported a vigorous acetylation of the p53 protein by nutlin-treatment. In this study we aimed to investigate the functional role of this p53 acetylation in nutlin-sensitivity, and further to explore if nutlin-induced protein acetylation in general could indicate novel targets for the enhancement of nutlin-based therapy. Results Nutlin-3 was found to enhance the acetylation of p53 in the human AML cell line MOLM-13 (wild type TP53) and in TP53 null cells transfected with wild type p53 cDNA. Stable isotope labeling with amino acids in cell culture (SILAC) in combination with immunoprecipitation using an anti-acetyl-lysine antibody and mass spectrometry analysis identified increased levels of acetylated Histone H2B, Hsp27 and Hsp90 in MOLM-13 cells after nutlin-treatment, accompanied by downregulation of total levels of Hsp27 and Hsp90. Intracellular levels of heat shock proteins Hsp27, Hsp40, Hsp60, Hsp70 and Hsp90α were correlated to nutlin-sensitivity for primary AML cells (n = 40), and AML patient samples with low sensitivity to nutlin-3 tended to express higher levels of heat shock proteins than more responsive samples. Combination therapy of nutlin-3 and Hsp90 inhibitor geldanamycin demonstrated synergistic induction of apoptosis in AML cell lines and primary AML cells. Finally, TP53 null cells transfected with a p53 acetylation defective mutant demonstrated decreased heat shock protein acetylation and sensitivity to nutlin-3 compared to wild type p53 expressing cells. Conclusions Altogether, our results demonstrate that nutlin-3 induces acetylation of p53, histones and heat shock proteins, and indicate that p53 acetylation status and the levels of heat shock proteins may participate in modulation of nutlin-3 sensitivity in AML

Disease-stabilizing treatment based on all-trans retinoic acid and valproic acid in acute myeloid leukemia – identification of responders by gene expression profiling of pretreatment leukemic cells

Abstract Background Acute myeloid leukemia (AML) is an aggressive malignancy only cured by intensive therapy. However, many elderly and unfit patients cannot receive such treatment due to an unacceptable risk of treatment-related morbidity and mortality. Disease-stabilizing therapy is then the only possible strategy, one alternative being treatment based on all-trans retinoic acid (ATRA) combined with the histone deacetylase inhibitor valproic acid and possibly low-toxicity conventional chemotherapy. Methods Primary AML cells were derived from 43 patients included in two clinical studies of treatment based on ATRA, valproic acid and theophyllamine; low toxicity chemotherapy (low-dose cytarabine, hydroxyurea, 6-mercaptopurin) was also allowed. Pretreatment leukemic cells were analyzed by mutation profiling of 54 genes frequently mutated in myeloid malignancies and by global gene expression profiling before and during in vivo treatment. Results Patients were classified as responders and non-responders to the treatment, however response to treatment showed no significant associations with karyotype or mutational profiles. Significance analysis of microarray (SAM) showed that responders and non-responders significantly differed with regard to the expression of 179 different genes. The differentially expressed genes encoding proteins with a known function were further classified based on the PANTHER (protein annotation through evolutionary relationship) classification system. The identified genes encoded proteins that are involved in several important biological functions, but a main subset of the genes were important for transcriptional regulation. These pretherapy differences in gene expression were largely maintained during treatment. Our analyses of primary AML cells during in vivo treatment suggest that ATRA modulates HOX activity (i.e. decreased expression of HOXA3, HOXA4 and HOXA5 and their regulator PBX3), but altered function of DNA methyl transferase 3A (DNMT3A) and G-protein coupled receptor signaling may also contribute to the effect of the overall treatment. Conclusions Responders and non-responders to AML stabilizing treatment based on ATRA and valproic acid differ in the pretreatment transcriptional regulation of their leukemic cells, and these differences may be important for the clinical effect of this treatment. Trial registrations ClinicalTrials.gov no. NCT00175812 ; EudraCT no. 2004–001663-22, registered September 9, 2005 and ClinicalTrials.gov no. NCT00995332 ; EudraCT no. 2007–2007–001995-36, registered October 14, 2009

Chronic Myeloid Leukemia Relapsing 25 Years after Allogenic Stem Cell Transplantation

Chronic myeloid leukemia (CML) is a myeloproliferative disorder in which neoplastic cells exhibit the Philadelphia chromosome and the related oncoproteinBCR-ABL1. Allogeneic stem cell transplantation (allo-SCT) was considered the first-line treatment for CML, before the introduction of tyrosine kinase inhibitors (TKIs). However, patients are at risk for relapse years after transplantation. We present a patient who relapsed 25 years after allo-SCT for chronic phase CML. Polymerase chain reaction (PCR) detected gradually evaluated levels ofBCR-ABL1transcripts, eventually leading to the diagnosis of relapsed disease. Additional mutational analyses did not reveal mutations in theBCR-ABL1gene, or other cooperating mutations. The patient was successfully treated with imatinib 400 mg daily, leading to new molecular remission. The case presentation emphasizes the need for long-term follow-up of such patients and the potential benefit of initiating TKI treatment with early signs of relapse

Chronic Myeloid Leukemia Relapsing 25 Years after Allogenic Stem Cell Transplantation

Chronic myeloid leukemia (CML) is a myeloproliferative disorder in which neoplastic cells exhibit the Philadelphia chromosome and the related oncoprotein BCR-ABL1. Allogeneic stem cell transplantation (allo-SCT) was considered the first-line treatment for CML, before the introduction of tyrosine kinase inhibitors (TKIs). However, patients are at risk for relapse years after transplantation. We present a patient who relapsed 25 years after allo-SCT for chronic phase CML. Polymerase chain reaction (PCR) detected gradually evaluated levels of BCR-ABL1 transcripts, eventually leading to the diagnosis of relapsed disease. Additional mutational analyses did not reveal mutations in the BCR-ABL1 gene, or other cooperating mutations. The patient was successfully treated with imatinib 400 mg daily, leading to new molecular remission. The case presentation emphasizes the need for long-term follow-up of such patients and the potential benefit of initiating TKI treatment with early signs of relapse

p53 Protein Isoform Profiles in AML: Correlation with Distinct Differentiation Stages and Response to Epigenetic Differentiation Therapy

p53 protein isoform expression has been found to correlate with prognosis and chemotherapy response in acute myeloid leukemia (AML). We aimed to investigate how p53 protein isoforms are modulated during epigenetic differentiation therapy in AML, and if p53 isoform expression could be a potential biomarker for predicting a response to this treatment. p53 full-length (FL), p53β and p53γ protein isoforms were analyzed by 1D and 2D gel immunoblots in AML cell lines, primary AML cells from untreated patients and AML cells from patients before and after treatment with valproic acid (VPA), all-trans retinoic acid (ATRA) and theophylline. Furthermore, global gene expression profiling analysis was performed on samples from the clinical protocol. Correlation analyses were performed between p53 protein isoform expression and in vitro VPA sensitivity and FAB (French–American–British) class in primary AML cells. The results show downregulation of p53β/γ and upregulation of p53FL in AML cell lines treated with VPA, and in some of the patients treated with differentiation therapy. p53FL positively correlated with in vitro VPA sensitivity and the FAB class of AML, while p53β/γ isoforms negatively correlated with the same. Our results indicate that p53 protein isoforms are modulated by and may predict sensitivity to differentiation therapy in AML

p53 Protein Isoform Profiles in AML: Correlation with Distinct Differentiation Stages and Response to Epigenetic Differentiation Therapy

p53 protein isoform expression has been found to correlate with prognosis and chemotherapy response in acute myeloid leukemia (AML). We aimed to investigate how p53 protein isoforms are modulated during epigenetic differentiation therapy in AML, and if p53 isoform expression could be a potential biomarker for predicting a response to this treatment. p53 full-length (FL), p53β and p53γ protein isoforms were analyzed by 1D and 2D gel immunoblots in AML cell lines, primary AML cells from untreated patients and AML cells from patients before and after treatment with valproic acid (VPA), all-trans retinoic acid (ATRA) and theophylline. Furthermore, global gene expression profiling analysis was performed on samples from the clinical protocol. Correlation analyses were performed between p53 protein isoform expression and in vitro VPA sensitivity and FAB (French–American–British) class in primary AML cells. The results show downregulation of p53β/γ and upregulation of p53FL in AML cell lines treated with VPA, and in some of the patients treated with differentiation therapy. p53FL positively correlated with in vitro VPA sensitivity and the FAB class of AML, while p53β/γ isoforms negatively correlated with the same. Our results indicate that p53 protein isoforms are modulated by and may predict sensitivity to differentiation therapy in AML