The vascular bone marrow niche influences outcome in chronic myeloid leukemia via the E-selectin - SCL/TAL1 - CD44 axis.

The endosteal bone marrow niche and vascular endothelial cells provide sanctuaries for leukemic cells. In murine chronic myeloid leukemia (CML) CD44 on leukemia cells and E-selectin on bone marrow endothelium are essential mediators for the engraftment of leukemic stem cells. We hypothesized that non-adhesion of CML-initiating cells to E-selectin on the bone marrow endothelium may lead to superior eradication of leukemic stem cells in CML after treatment with imatinib than imatinib alone. Indeed, here we show that treatment with the E-selectin inhibitor GMI-1271 in combination with imatinib prolongs survival of mice with CML via decreased contact time of leukemia cells with bone marrow endothelium. Non-adhesion of BCR-ABL1+ cells leads to an increase of cell cycle progression and an increase of expression of the hematopoietic transcription factor and proto-oncogene Scl/Tal1 in leukemia-initiating cells. We implicate SCL/TAL1 as an indirect phosphorylation target of BCR-ABL1 and as a negative transcriptional regulator of CD44 expression. We show that increased SCL/TAL1 expression is associated with improved outcome in human CML. These data demonstrate the BCR-ABL1-specific, cell-intrinsic pathways leading to altered interactions with the vascular niche via the modulation of adhesion molecules - which could be exploited therapeutically in the future.

RNA Therapeutics in Cardiovascular Disease.

Noncoding RNAs have been shown to exert important physiological and pathophysiological functions. Various studies suggest that modulating noncoding RNAs may provide a therapeutic option. Noncoding RNAs comprise small RNAs, mainly microRNAs, and long noncoding RNAs. MicroRNAs postranscriptionally regulate gene expression pattern by binding to the 3'untranslated region of a given target mRNA, thereby blocking protein translation or inducing its degradation. Long noncoding RNAs on the contrary have more diverse functions acting as epigenetic regulators, molecular scaffolds, or decoys. In this article, we summarize examples of microRNAs and long noncoding RNAs, which might be promising novel targets for treatment of cardiovascular diseases, such as heart failure, acute myocardial infarction, fibrosis, as well as atherosclerosis. Furthermore, we give insights into the available tools to inhibit or overexpress noncoding RNAs and discuss the challenges for translation. Strategies for improving RNA therapeutics and reducing toxicity, for example, by augmenting tissue specificity or cellular uptake will be discussed.

Clonal Expansion of Endothelial Cells Contributes to Ischemia-Induced Neovascularization.

RATIONALE: Vascularization is critical to maintain organ function. Although many molecular pathways were shown to control vessel growth, the genuine process of capillary formation under different conditions is unclear. OBJECTIVE: Here, we elucidated whether clonal expansion contributes to vessel growth by using Confetti mice for genetic tracing of clonally expanding endothelial cells (ECs). METHODS AND RESULTS: In postnatal retina angiogenesis, we predominantly observed random distribution of fluorescence labeled ECs indicative of random integration or cell mixing. However, in models of pathophysiological angiogenesis (retinopathy of prematurity), as well as ischemia-induced angiogenesis in limbs and hearts, clonally expanded ECs were significantly more abundant (≤69%). Inhibition of VEGFR2 (vascular endothelial growth factor receptor 2) reduced clonal expansion after ischemia. To determine the mechanism underlying clonal expansion in vivo, we assessed gene expression specifically in clonally expanded ECs selected by laser capture microscopy. Clonally expanded ECs showed an enrichment of genes involved in endothelial-to-mesenchymal transition. Moreover, hypoxia-induced clonal expansion and endothelial-to-mesenchymal transition in ECs in vitro suggesting that hypoxia-enhanced endothelial-to-mesenchymal transition might contribute to vessel growth under ischemia. CONCLUSIONS: Our data suggest that neovascularization after ischemia is partially mediated by clonal expansion of ECs. Identification of the pathways that control clonal expansion may provide novel tools to augment therapeutic neovascularization or treat pathological angiogenesis.

JMJD8 Regulates Angiogenic Sprouting and Cellular Metabolism by Interacting With Pyruvate Kinase M2 in Endothelial Cells.

OBJECTIVE: Jumonji C (JmjC) domain-containing proteins modify histone and nonhistone proteins thereby controlling cellular functions. However, the role of JmjC proteins in angiogenesis is largely unknown. Here, we characterize the expression of JmjC domain-containing proteins after inducing endothelial differentiation of murine embryonic stem cells and study the function of JmjC domain-only proteins in endothelial cell (EC) functions. APPROACH AND RESULTS: We identified a large number of JmjC domain-containing proteins regulated by endothelial differentiation of murine embryonic stem cells. Among the family of JmjC domain-only proteins, Jmjd8 was significantly upregulated on endothelial differentiation. Knockdown of Jmjd8 in ECs significantly decreased in vitro network formation and sprouting in the spheroid assay. JMJD8 is exclusively detectable in the cytoplasm, excluding a function as a histone-modifying enzyme. Mass spectrometry analysis revealed JMJD8-interacting proteins with known functions in cellular metabolism like pyruvate kinase M2. Accordingly, knockdown of pyruvate kinase M2 in human umbilical vein ECs decreased endothelial sprouting in the spheroid assay. Knockdown of JMJD8 caused a reduction of EC metabolism as measured by Seahorse Bioscience extracellular flux analysis. Conversely, overexpression of JMJD8 enhanced cellular oxygen consumption rate of ECs, reflecting an increased mitochondrial respiration. CONCLUSIONS: Jmjd8 is upregulated during endothelial differentiation and regulates endothelial sprouting and metabolism by interacting with pyruvate kinase M2.

Laminar shear stress inhibits endothelial cell metabolism via KLF2-mediated repression of PFKFB3.

OBJECTIVE: Cellular metabolism was recently shown to regulate endothelial cell phenotype profoundly. Whether the atheroprotective biomechanical stimulus elicited by laminar shear stress modulates endothelial cell metabolism is not known. APPROACH AND RESULTS: Here, we show that laminar flow exposure reduced glucose uptake and mitochondrial content in endothelium. Shear stress-mediated reduction of endothelial metabolism was reversed by silencing the flow-sensitive transcription factor Krüppel-like factor 2 (KLF2). Endothelial-specific deletion of KLF2 in mice induced glucose uptake in endothelial cells of perfused hearts. KLF2 overexpression recapitulates the inhibitory effects on endothelial glycolysis elicited by laminar flow, as measured by Seahorse flux analysis and glucose uptake measurements. RNA sequencing showed that shear stress reduced the expression of key glycolytic enzymes, such as 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-3 (PFKFB3), phosphofructokinase-1, and hexokinase 2 in a KLF2-dependent manner. Moreover, KLF2 represses PFKFB3 promoter activity. PFKFB3 knockdown reduced glycolysis, and overexpression increased glycolysis and partially reversed the KLF2-mediated reduction in glycolysis. Furthermore, PFKFB3 overexpression reversed KLF2-mediated reduction in angiogenic sprouting and network formation. CONCLUSIONS: Our data demonstrate that shear stress-mediated repression of endothelial cell metabolism via KLF2 and PFKFB3 controls endothelial cell phenotype.

CCR2 recruits an inflammatory macrophage subpopulation critical for angiogenesis in tissue repair.

Monocytes/macrophages are critical in orchestrating the tissue-repair response. However, the mechanisms that govern macrophage regenerative activities during the sequential phases of repair are largely unknown. In the present study, we examined the dynamics and functions of diverse monocyte/macrophage phenotypes during the sequential stages of skin repair. By combining the analysis of a new CCR2-eGFP reporter mouse model with conditional mouse mutants defective in myeloid cell-restricted CCR2 signaling or VEGF-A synthesis, we show herein that among the large number of inflammatory CCR2(+)Ly6C(+) macrophages that dominate the early stage of repair, only a small fraction strongly expresses VEGF-A that has nonredundant functions for the induction of vascular sprouts. The switch of macrophage-derived VEGF-A during the early stage of tissue growth toward epidermal-derived VEGF-A during the late stage of tissue maturation was critical to achieving physiologic tissue vascularization and healing progression. The results of the present study provide new mechanistic insights into CCR2-mediated recruitment of blood monocyte subsets into damaged tissue, the dynamics and functional consequences of macrophage plasticity during the sequential repair phases, and the complementary role of macrophage-derived VEGF-A in coordinating effective tissue growth and vascularization in the context of tissue-resident wound cells. Our findings may be relevant for novel monocyte-based therapies to promote tissue vascularization.

The Walter Reed Project, Kisumu Field Station: Impact of Research on Malaria Policy, Management, and Prevention.

The Walter Reed Project is a collaboration between the Walter Reed Army Institute of Research of the United States Department of Defense and the Kenya Medical Research Institute. The Kisumu field station, comprising four campuses, has until recently been devoted primarily to research on malaria countermeasures. The Kombewa Clinical Research Center is dedicated to conducting regulated clinical trials of therapeutic and vaccine candidates in development. The center's robust population-based surveillance platform, along with an active community engagement strategy, guarantees consistent recruitment and retention of participants in clinical trials. The Malaria Diagnostic Center, backed by WHO-certified microscopists and a large malaria blood film collection, champions high-quality malaria diagnosis and strict quality assurance through standardized microscopy trainings. The Malaria Drug Resistance Laboratory leverages cutting-edge technology such as real-time Polymerase Chain Reaction (qPCR) to conduct comprehensive research on resistance markers and obtain information on drug efficacy. The laboratory has been working on validating artemisinin resistance markers and improving tracking methods for current and future antimalarial compounds. Finally, the Basic Science Laboratory employs advanced genomic technology to examine endpoints such as immunogenicity and genomic fingerprinting for candidate drugs and vaccine efficacy. Herein, we examine the site's significant contributions to malaria policy, management, and prevention practices in Kenya and around the world.

Deployment of Rotavirus Vaccine in Western Kenya Coincides with a Reduction in All-Cause Child Mortality: A Retrospective Cohort Study.

Rotavirus is an important cause of fatal pediatric diarrhea worldwide. Many national immunization programs began adding rotavirus vaccine following a 2009 World Health Organization recommendation. Kenya added rotavirus vaccine to their immunization program at the end of 2014. From a cohort of 38,463 children in the Kisumu health and demographic surveillance site in western Kenya, we assessed how the implementation of the rotavirus vaccine affected mortality in children under 3 years of age. Following its introduction in late 2014, the span of rotavirus vaccine coverage for children increased to 75% by 2017. Receiving the rotavirus vaccine was associated with a 44% reduction in all-cause child mortality (95% confidence interval = 28-68%, p < 0.0001), but not diarrhea-specific mortality (p = 0.401). All-cause child mortality declined 2% per month following the implementation of the rotavirus vaccine (p = 0.002) among both vaccinated and unvaccinated children, but diarrhea-specific mortality was not associated with the implementation of the rotavirus vaccine independent of individual vaccine status (p = 0.125). The incidence of acute diarrhea decreased over the study period, and the introduction of the rotavirus vaccine was not associated with population-wide trends (p = 0.452). The receipt of the rotavirus vaccine was associated with a 34% reduction in the incidence of diarrhea (95% confidence interval = 24-43% reduction). These results suggest that rotavirus vaccine may have had an impact on all-cause child mortality. The analyses of diarrhea-specific mortality were limited by relatively few deaths (n = 57), as others have found a strong reduction in diarrhea-specific mortality. Selection bias may have played a part in these results-children receiving rotavirus vaccine were more likely to be fully immunized than children not receiving the rotavirus vaccine.

Differential roles of macrophages in diverse phases of skin repair.

Influx of macrophages plays a crucial role in tissue repair. However, the precise function of macrophages during the healing response has remained a subject of debate due to their functional dichotomy as effectors of both tissue injury and repair. We tested the hypothesis that macrophages recruited during the diverse phases of skin repair after mechanical injury exert specific functions to restore tissue integrity. For this purpose, we developed a mouse model that allows conditional depletion of macrophages during the sequential stages of the repair response. Depletion of macrophages restricted to the early stage of the repair response (inflammatory phase) significantly reduced the formation of vascularized granulation tissue, impaired epithelialization, and resulted in minimized scar formation. In contrast, depletion of macrophages restricted to the consecutive mid-stage of the repair response (phase of tissue formation) resulted in severe hemorrhage in the wound tissue. Under these conditions, transition into the subsequent phase of tissue maturation and wound closure did not occur. Finally, macrophage depletion restricted to the late stage of repair (phase of tissue maturation) did not significantly impact the outcome of the repair response. These results demonstrate that macrophages exert distinct functions during the diverse phases of skin repair, which are crucial to control the natural sequence of repair events.

Simultaneous fitting of real-time PCR data with efficiency of amplification modeled as Gaussian function of target fluorescence.

BACKGROUND: In real-time PCR, it is necessary to consider the efficiency of amplification (EA) of amplicons in order to determine initial target levels properly. EAs can be deduced from standard curves, but these involve extra effort and cost and may yield invalid EAs. Alternatively, EA can be extracted from individual fluorescence curves. Unfortunately, this is not reliable enough. RESULTS: Here we introduce simultaneous non-linear fitting to determine - without standard curves - an optimal common EA for all samples of a group. In order to adjust EA as a function of target fluorescence, and still to describe fluorescence as a function of cycle number, we use an iterative algorithm that increases fluorescence cycle by cycle and thus simulates the PCR process. A Gauss peak function is used to model the decrease of EA with increasing amplicon accumulation. Our approach was validated experimentally with hydrolysis probe or SYBR green detection with dilution series of 5 different targets. It performed distinctly better in terms of accuracy than standard curve, DART-PCR, and LinRegPCR approaches. Based on reliable EAs, it was possible to detect that for some amplicons, extraordinary fluorescence (EA > 2.00) was generated with locked nucleic acid hydrolysis probes, but not with SYBR green. CONCLUSION: In comparison to previously reported approaches that are based on the separate analysis of each curve and on modelling EA as a function of cycle number, our approach yields more accurate and precise estimates of relative initial target levels.

Myeloid Kdm6b deficiency results in advanced atherosclerosis.

BACKGROUND AND AIMS: Atherosclerosis is a lipid-driven chronic inflammatory disorder of the arteries, and monocytes and macrophages play a central role in this process. Within the atherosclerotic lesion, macrophages can scavenge modified lipids and become the so-called foam cells. We previously reported that the epigenetic enzyme Kdm6b (also known as Jmjd3) controls the pro-fibrotic transcriptional profile of peritoneal foam cells. Given the importance of these cells in atherosclerosis, we now studied the effect of myeloid Kdm6b on disease progression. METHODS: Bone marrow of myeloid Kdm6b deficient (Kdm6bdel) mice or wild type littermates (Kdm6bwt) was transplanted to lethally irradiated Ldlr-/- mice fed a high fat diet for 9 weeks to induce atherosclerosis. RESULTS: Lesion size was similar in Kdm6bwt and Kdm6bdel transplanted mice. However, lesions of Kdm6bdel mice contained more collagen and were more necrotic. Pathway analysis on peritoneal foam cells showed that the pathway involved in leukocyte chemotaxis was most significantly upregulated. Although macrophage and neutrophil content was similar after 9 weeks of high fat diet feeding, the relative increase in collagen content and necrosis revealed that atherosclerotic lesions in Kdm6bdel mice progress faster. CONCLUSION: Myeloid Kdm6b deficiency results in more advanced atherosclerosis.

Light-inducible antimiR-92a as a therapeutic strategy to promote skin repair in healing-impaired diabetic mice.

MicroRNAs (miRs) are small non-coding RNAs that post-transcriptionally control gene expression. Inhibition of miRs by antisense RNAs (antimiRs) might be a therapeutic option for many diseases, but systemic inhibition can have adverse effects. Here we show that light-activatable antimiRs efficiently and locally restricted target miR activity in vivo. We use an antimiR-92a and establish a therapeutic benefit in diabetic wound healing. AntimiR-92a is modified with photolabile protecting groups, so called 'cages'. Irradiation activates intradermally injected caged antimiR-92a without substantially affecting miR-92a expression in other organs. Light activation of caged antimiR-92a improves healing in diabetic mice to a similar extent as conventional antimiRs and derepresses the miR-92a targets Itga5 and Sirt1, thereby regulating wound cell proliferation and angiogenesis. These data show that light can be used to locally activate therapeutically active antimiRs in vivo.

Macrophage Kdm6b controls the pro-fibrotic transcriptome signature of foam cells.

AIM: In order to identify regulators of foam cells, we studied the H3K27 demethylase Kdm6b (also known as Jmjd3), a known regulator of macrophages, in controlling the transcriptional profile of foam cells. MATERIALS & METHODS: Foam cells from Kdm6b-deleted or Kdm6b wild-type mice were isolated and used for RNA-sequencing analysis. RESULTS: Pathway analysis revealed that pro-fibrotic pathways were strongly suppressed in Kdm6b-deleted foam cells. Analysis of published datasets showed that foam cell formation induces these pro-fibrotic characteristics. Overlay of both datasets indicated that fibrotic genes which are induced upon foam cell formation, are reduced in the absence of Kdm6b. These data suggest that foam cell formation induces a pro-fibrotic gene signature in a Kdm6b-dependent manner. CONCLUSION: We identified Kdm6b as a novel regulator of the pro-fibrotic signature of peritoneal foam cells.

Shear stress-regulated miR-27b controls pericyte recruitment by repressing SEMA6A and SEMA6D.

AIMS: Vessel maturation involves the recruitment of mural cells such as pericytes and smooth muscle cells. Laminar shear stress is a major trigger for vessel maturation, but the molecular mechanisms by which shear stress affects recruitment of pericytes are unclear. MicroRNAs (miRs) are small non-coding RNAs, which post-transcriptionally control gene expression. The aim of the present study was to unveil the mechanism by which shear stress-regulated microRNAs contribute to vessel maturation. METHODS AND RESULTS: Here, we show that laminar shear stress increased miR-27a and miR-27b expression in vitro and in ex vivo in mouse femoral artery explants. Overexpression of miR-27b in endothelial cells increased pericyte adhesion and pericyte recruitment in vitro. In vitro barrier function of endothelial-pericyte co-cultures was augmented by miR-27b overexpression, whereas inhibition of miR-27a/b reduced adhesion and pericyte coverage and decreased barrier functions. In vivo, pharmacological inhibition of miR-27a/b by locked nucleic acid antisense oligonucleotides significantly reduced pericyte coverage and increased water content in the murine uterus. MiR-27b overexpression repressed semaphorins (SEMA), which mediate repulsive signals, and the vessel destabilizing human but not mouse Angiopoietin-2 (Ang-2). Silencing of SEMA6A and SEMA6D rescued the reduced pericyte adhesion by miR-27 inhibition. Furthermore, inhibition of SEMA6D increased barrier function of an endothelial-pericyte co-culture in vitro. CONCLUSION: The present study demonstrates for the first time that shear stress-regulated miR-27b promotes the interaction of endothelial cells with pericytes, partly by repressing SEMA6A and SEMA6D.

Distinct functions of epidermal and myeloid-derived VEGF-A in skin tumorigenesis mediated by HPV8.

Beta human papillomaviruses (HPV) have been suspected to be carcinogenic in nonmelanoma skin cancers (NMSC), but the basis for potential viral contributions to these cancers is poorly understood. In particular, it is unresolved how HPV-infected keratinocytes escape cell-cycle control and whether their cross-talk with immune cells is critical for tumorigenesis. In nonviral preclinical models, the angiogenic cytokine VEGF-A has been identified as a critical regulator of NMSC. In this study, we dissected the contribution of epidermal versus myeloid cell-derived VEGF-A in HPV-mediated skin cancer by interbreeding an HPV8 transgenic mouse model with a conditional disruption of VEGF-A restricted to either epidermal or myeloid cells. Although only epidermal-derived VEGF-A was essential for initiation of skin tumor development, both spontaneously and UV-light triggered, both epidermal and myeloid cell-derived VEGF-A contributed to regeneration-induced tumorigenesis upon HPV8 overexpression, partly not only through a paracrine effect on endothelial cells, but also most probably through an additional autocrine effect on epidermal cells. Our findings offer new mechanistic insights into distinct functions of epidermal versus myeloid cell-derived VEGF-A during HPV-mediated tumorigenesis, with possible implications for preventing this disease.