Megakaryocyte Cytoskeletal Proteins in Platelet Biogenesis and Diseases

Thrombopoiesis governs the formation of blood platelets in bone marrow by converting megakaryocytes into long, branched proplatelets on which individual platelets are assembled. The megakaryocyte cytoskeleton responds to multiple microenvironmental cues, including chemical and mechanical stimuli, sustaining the platelet shedding. During the megakaryocyte's life cycle, cytoskeletal networks organize cell shape and content, connect them physically and biochemically to the bone marrow vascular niche, and enable the release of platelets into the bloodstream. While the basic building blocks of the cytoskeleton have been studied extensively, new sets of cytoskeleton regulators have emerged as critical components of the dynamic protein network that supports platelet production. Understanding how the interaction of individual molecules of the cytoskeleton governs megakaryocyte behavior is essential to improve knowledge of platelet biogenesis and develop new therapeutic strategies for inherited thrombocytopenias caused by alterations in the cytoskeletal genes.

Bioengineering the Bone Marrow Vascular Niche

The bone marrow (BM) tissue is the main physiological site for adult hematopoiesis. In recent years, the cellular and matrix components composing the BM have been defined with unprecedent resolution, both at the molecular and structural levels. With the expansion of this knowledge, the possibility of reproducing a BM-like structure, to ectopically support and study hematopoiesis, becomes a reality. A number of experimental systems have been implemented and have displayed the feasibility of bioengineering BM tissues, supported by cells of mesenchymal origin. Despite being known as an abundant component of the BM, the vasculature has been largely disregarded for its role in regulating tissue formation, organization and determination. Recent reports have highlighted the crucial role for vascular endothelial cells in shaping tissue development and supporting steady state, emergency and malignant hematopoiesis, both pre- and postnatally. Herein, we review the field of BM-tissue bioengineering with a particular focus on vascular system implementation and integration, starting from describing a variety of applicable in vitro models, ending up with in vivo preclinical models. Additionally, we highlight the challenges of the field and discuss the clinical perspectives in terms of adoptive transfer of vascularized BM-niche grafts in patients to support recovering hematopoiesis.

Endothelial Jak3 expression enhances pro-hematopoietic angiocrine function in mice

Jak3 is the only non-promiscuous member of the Jak family of secondary messengers. Studies to date have focused on understanding and targeting the cell-autonomous role of Jak3 in immunity, while functional Jak3 expression outside the hematopoietic system remains largely unreported. We show that Jak3 is expressed in endothelial cells across hematopoietic and non-hematopoietic organs, with heightened expression in the bone marrow. The bone marrow niche is understood as a network of different cell types that regulate hematopoietic function. We show that the Jak3–/– bone marrow niche is deleterious for the maintenance of long-term repopulating hematopoietic stem cells (LT-HSCs) and that JAK3-overexpressing endothelial cells have increased potential to expand LT-HSCs in vitro. This work may serve to identify a novel function for a highly specific tyrosine kinase in the bone marrow vascular niche and to further characterize the LT-HSC function of sinusoidal endothelium.

The Dynamic Interface Between the Bone Marrow Vascular Niche and Hematopoietic Stem Cells in Myeloid Malignancy

Hematopoietic stem cells interact with bone marrow niches, including highly specialized blood vessels. Recent studies have revealed the phenotypic and functional heterogeneity of bone marrow endothelial cells. This has facilitated the analysis of the vascular microenvironment in steady state and malignant hematopoiesis. In this review, we provide an overview of the bone marrow microenvironment, focusing on refined analyses of the marrow vascular compartment performed in mouse studies. We also discuss the emerging role of the vascular niche in “inflamm-aging” and clonal hematopoiesis, and how the endothelial microenvironment influences, supports and interacts with hematopoietic cells in acute myeloid leukemia and myelodysplastic syndromes, as exemplar states of malignant myelopoiesis. Finally, we provide an overview of strategies for modulating these bidirectional interactions to therapeutic effect in myeloid malignancies.

6091Chronic heart failure is associated with inflammatory deterioration of the bone marrow vascular niche

Introduction Inflammation plays a crucial role in many aspects of cardiovascular disease. Particularly, acquired mutations of hematopoietic stem cells (HSC) leading to clonal expansion of inflammatory cells (CHIP) are increased with age and are associated with an enhanced risk of cardiovascular disease. The bone marrow (BM) vascular niche plays a crucial role in maintenance and regulation of HSC functions. Previous studies in mice showed the reduction of a specific Endomucin-high (H-type) endothelial cells (EC) subpopulation by aging. However, the impact of cardiovascular disease is unclear. Therefore, we aimed to investigate the effects of age and heart failure (HF) on the vascular BM cell composition in mice and humans. Methods and results Aging mice showed an age-dependent decrease of type H (Emcn-high) BM ECs (p=0.004), whereas the BM frequencies of type L (Emcn-low) ECs did not significantly differ (P=0.18). Importantly, we also observed a marked reduction of type H EC in chronic ischemic mice (P=0.016 vs. sham) indicating that chronic ischemic HF induces similar alterations of the vascular stem cell niche. Importantly, type H ECs were also significantly reduced in ischemic HF patients (n=16) compared with control subjects (n=8; P=0.0003). To gain insights into the mechanisms underlying the changes in the vascular niche, we performed single cells RNA sequencing of human BM ECs. These studies confirmed the decrease in Emcn-expressing ECs in ischemic HF patients, which was accompanied by significantly increased expression of inflammatory genes, including IL1b (P<0.0001 vs. control). Inflammatory EC phenotypes and IL1b expression in HF could be further confirmed at protein level using cytospin immunostainings. Finally, we comprehensively evaluated phenotype-associated differences in the bone marrow plasma proteomes of healthy individuals (n=19) and patients with chronic ischemic (n=22) and non-ischemic (n=19) HF, using proximity extension assays. Here, we identified 182 proteins significantly differentially regulated in CHF versus CTRL. Among the top upregulated proteins the BM environment of patients with CHF showed a striking enrichment of inflammatory and ECM remodeling components. Conclusions Our data show for the first time an impact of chronic heart failure on the bone marrow vascular niche in humans. These changes seem to be strongly associated with increased inflammatory response and bone matrix remodeling in CHF. Specifically, the induction of the inflammatory cytokine IL1b may contribute to the disturbed phenotype suggesting that inhibition of IL1b (e.g. by canakinumab) may be used as a novel strategy to prevent or reverse the deterioration of the vascular BM niche.

RUNX transcription factors potentially control E-selectin expression in the bone marrow vascular niche in mice

Key Points RUNX transcription factors potentially transactivate E-selectin expression in the vascular niche. RUNX inhibitor disrupts engraftment of AML cells in the bone marrow, possibly by attenuating E-selectin expression.

Interferon Alpha Mediated Activation of the Bone Marrow Stem Cell Vascular Niche

Endothelial cells (ECs) significantly influence the response of an organism to inflammation and infection. In the bone marrow, these cells form a major part of the bone marrow vascular niche, which regulates stem cell function and influences stem cell fate. The primary response to infection involves synthesis of immune-modulatory cytokines, such as interferon alpha (IFNα). We, and others, have shown that in contrast to the anti-proliferative effect of IFNα on hematopoietic stem cells (HSCs) in vitro, in vivo, IFNα induces cell cycle entry of quiescent HSCs (Essers et al. 2009). Given the contrasting outcome of in vitro and in vivo exposure of HSCs to IFNα, it is probable that niche cells and molecular maintenance signals from the niche are required for IFNα-induced activation of quiescent HSCs. Here, we now show that although interferon signaling itself in niche cells is not required for HSC activation, niche components do respond to IFNα stimulation. Of these, bone marrow ECs are rapidly and indirectly stimulated following IFNα treatment in vivo. The vascular system, lined by ECs, is a central primary responder following inflammatory insult with a multifaceted role, including transport of immune cells and promotion of a rapid return to homeostasis. We have found that IFNα stimulation in vivo results in an increased bone marrow vascularity, visualized by fluorescence imaging of cryo-sectioned murine femurs immunostained with the vascular basement membrane protein, laminin. IFNα-mediated activation of ECs involves the expression of key inflammatory and endothelial-stimulatory markers, including VE-cadherin and ESAM, and also an increased vascular leakiness in the bone marrow, demonstrated by the Evans blue assay. In accordance with this finding of vascular activation, we confirmed that VEGF, which is an established regulator of vascular dynamics, is rapidly up regulated in the bone marrow supernatant of treated mice. Furthermore, we can also demonstrate a key role for VEGF in our observed IFNα-mediated stimulation of ECs by abrogation of this activation using co-treatment with Avastin (bevacizumab) in vivo. We are now investigating the feedback from this activation of ECs on the hematopoietic system itself. In summary, these data indicate a rapid and indirect stimulation of the bone marrow vascular niche in an inflammatory setting. In addition, they support a previously undescribed cellular instruction from an activated hematopoietic system to a niche component. Disclosures No relevant conflicts of interest to declare.