Author Correction: The bone marrow microenvironment at single-cell resolution.

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The bone marrow microenvironment at single-cell resolution.

The bone marrow microenvironment has a key role in regulating haematopoiesis, but its molecular complexity and response to stress are incompletely understood. Here we map the transcriptional landscape of mouse bone marrow vascular, perivascular and osteoblast cell populations at single-cell resolution, both at homeostasis and under conditions of stress-induced haematopoiesis. This analysis revealed previously unappreciated levels of cellular heterogeneity within the bone marrow niche and resolved cellular sources of pro-haematopoietic growth factors, chemokines and membrane-bound ligands. Our studies demonstrate a considerable transcriptional remodelling of niche elements under stress conditions, including an adipocytic skewing of perivascular cells. Among the stress-induced changes, we observed that vascular Notch delta-like ligands (encoded by Dll1 and Dll4) were downregulated. In the absence of vascular Dll4, haematopoietic stem cells prematurely induced a myeloid transcriptional program. These findings refine our understanding of the cellular architecture of the bone marrow niche, reveal a dynamic and heterogeneous molecular landscape that is highly sensitive to stress and illustrate the utility of single-cell transcriptomic data in evaluating the regulation of haematopoiesis by discrete niche populations.

Mutations in kelch-like 3 and cullin 3 cause hypertension and electrolyte abnormalities.

Hypertension affects one billion people and is a principal reversible risk factor for cardiovascular disease. Pseudohypoaldosteronism type II (PHAII), a rare Mendelian syndrome featuring hypertension, hyperkalaemia and metabolic acidosis, has revealed previously unrecognized physiology orchestrating the balance between renal salt reabsorption and K(+) and H(+) excretion. Here we used exome sequencing to identify mutations in kelch-like 3 (KLHL3) or cullin 3 (CUL3) in PHAII patients from 41 unrelated families. KLHL3 mutations are either recessive or dominant, whereas CUL3 mutations are dominant and predominantly de novo. CUL3 and BTB-domain-containing kelch proteins such as KLHL3 are components of cullin-RING E3 ligase complexes that ubiquitinate substrates bound to kelch propeller domains. Dominant KLHL3 mutations are clustered in short segments within the kelch propeller and BTB domains implicated in substrate and cullin binding, respectively. Diverse CUL3 mutations all result in skipping of exon 9, producing an in-frame deletion. Because dominant KLHL3 and CUL3 mutations both phenocopy recessive loss-of-function KLHL3 mutations, they may abrogate ubiquitination of KLHL3 substrates. Disease features are reversed by thiazide diuretics, which inhibit the Na-Cl cotransporter in the distal nephron of the kidney; KLHL3 and CUL3 are expressed in this location, suggesting a mechanistic link between KLHL3 and CUL3 mutations, increased Na-Cl reabsorption, and disease pathogenesis. These findings demonstrate the utility of exome sequencing in disease gene identification despite the combined complexities of locus heterogeneity, mixed models of transmission and frequent de novo mutation, and establish a fundamental role for KLHL3 and CUL3 in blood pressure, K(+) and pH homeostasis.

Complementary and Inducible creERT2 Mouse Models for Functional Evaluation of Endothelial Cell Subtypes in the Bone Marrow.

In the adult bone marrow (BM), endothelial cells (ECs) are an integral component of the hematopoietic stem cell (HSC)-supportive niche, which modulates HSC activity by producing secreted and membrane-bound paracrine signals. Within the BM, distinct vascular arteriole, transitional, and sinusoidal EC subtypes display unique paracrine expression profiles and create anatomically-discrete microenvironments. However, the relative contributions of vascular endothelial subtypes in supporting hematopoiesis is unclear. Moreover, constitutive expression and off-target activity of currently available endothelial-specific and endothelial-subtype-specific murine cre lines potentially confound data analysis and interpretation. To address this, we describe two tamoxifen-inducible cre-expressing lines, Vegfr3-creERT2 and Cx40-creERT2, that efficiently label sinusoidal/transitional and arteriole endothelium respectively in adult marrow, without off-target activity in hematopoietic or perivascular cells. Utilizing an established mouse model in which cre-dependent recombination constitutively-activates MAPK signaling within adult endothelium, we identify arteriole ECs as the driver of MAPK-mediated hematopoietic dysfunction. These results define complementary tamoxifen-inducible creERT2-expressing mouse lines that label functionally-discrete and non-overlapping sinusoidal/transitional and arteriole EC populations in the adult BM, providing a robust toolset to investigate the differential contributions of vascular subtypes in maintaining hematopoietic homeostasis.

Restoring bone marrow niche function rejuvenates aged hematopoietic stem cells by reactivating the DNA Damage Response.

Aging associated defects within stem cell-supportive niches contribute towards age-related decline in stem cell activity. However, mechanisms underlying age-related niche defects, and whether restoring niche function can improve stem cell fitness, remain unclear. Here, we sought to determine whether aged blood stem cell function can be restored by rejuvenating their supportive niches within the bone marrow (BM). We identify Netrin-1 as a critical regulator of BM niche cell aging. Niche-specific deletion of Netrin-1 induces premature aging phenotypes within the BM microenvironment, while supplementation of aged mice with Netrin-1 rejuvenates aged niche cells and restores competitive fitness of aged blood stem cells to youthful levels. We show that Netrin-1 plays an essential role in maintaining active DNA damage responses (DDR), and that aging-associated decline in niche-derived Netrin-1 results in DNA damage accumulation within the BM microenvironment. We show that Netrin-1 supplementation is sufficient to resolve DNA damage and restore regenerative potential of the aged BM niche and blood stem cells to endure serial chemotherapy regimens.

Influence of Decreased Temperature of Tensile Testing on the Annealing-Induced Hardening and Deformation-Induced Softening Effects in Ultrafine-Grained Al-0.4Zr Alloy.

The influence of decreased temperature of tensile testing on annealing-induced hardening (AIH) and deformation-induced softening (DIS) effects has been studied in an ultrafine-grained (UFG) Al-Zr alloy produced by high-pressure torsion. We show that the UFG Al-Zr alloy demonstrates a DIS effect accompanied by a substantial increase in the elongation to failure δ (up to δ ≈ 30%) depending on the value of additional straining. Both the AIH and DIS effects weaken with a decrease in the tensile test temperature. The critical deformation temperatures were revealed at which the AIH and DIS effects are suppressed. The activation energy Q of plastic flow has been estimated for the UFG Al-Zr alloy in the as-processed, subsequently annealed and additionally strained states. It was shown that the annealing decreases the Q-value from ~80 kJ/mol to 23-28 kJ/mol, while the subsequent additional straining restores the initial Q-value. Alloying with Zr results in the expansion of the temperature range of the AIH effect manifestation to lower temperatures and results in the change in the Q-value in all of the studied states compared to the HPT-processed Al. The obtained Q-values and underlying flow mechanisms are discussed in correlation with specific microstructural features and in comparison to the UFG Al.

Endothelial mTOR maintains hematopoiesis during aging.

Aging leads to a decline in hematopoietic stem and progenitor cell (HSPC) function. We recently discovered that aging of bone marrow endothelial cells (BMECs) leads to an altered crosstalk between the BMEC niche and HSPCs, which instructs young HSPCs to behave as aged HSPCs. Here, we demonstrate aging leads to a decrease in mTOR signaling within BMECs that potentially underlies the age-related impairment of their niche activity. Our findings reveal that pharmacological inhibition of mTOR using Rapamycin has deleterious effects on hematopoiesis. To formally determine whether endothelial-specific inhibition of mTOR can influence hematopoietic aging, we conditionally deleted mTOR in ECs (mTOR(ECKO)) of young mice and observed that their HSPCs displayed attributes of an aged hematopoietic system. Transcriptional profiling of HSPCs from mTOR(ECKO) mice revealed that their transcriptome resembled aged HSPCs. Notably, during serial transplantations, exposure of wild-type HSPCs to an mTOR(ECKO) microenvironment was sufficient to recapitulate aging-associated phenotypes, confirming the instructive role of EC-derived signals in governing HSPC aging.

Chronic activation of endothelial MAPK disrupts hematopoiesis via NFKB dependent inflammatory stress reversible by SCGF.

Inflammatory signals arising from the microenvironment have emerged as critical regulators of hematopoietic stem cell (HSC) function during diverse processes including embryonic development, infectious diseases, and myelosuppressive injuries caused by irradiation and chemotherapy. However, the contributions of cellular subsets within the microenvironment that elicit niche-driven inflammation remain poorly understood. Here, we identify endothelial cells as a crucial component in driving bone marrow (BM) inflammation and HSC dysfunction observed following myelosuppression. We demonstrate that sustained activation of endothelial MAPK causes NF-κB-dependent inflammatory stress response within the BM, leading to significant HSC dysfunction including loss of engraftment ability and a myeloid-biased output. These phenotypes are resolved upon inhibition of endothelial NF-κB signaling. We identify SCGF as a niche-derived factor that suppresses BM inflammation and enhances hematopoietic recovery following myelosuppression. Our findings demonstrate that chronic endothelial inflammation adversely impacts niche activity and HSC function which is reversible upon suppression of inflammation.

Endothelial transplantation rejuvenates aged hematopoietic stem cell function.

Age-related changes in the hematopoietic compartment are primarily attributed to cell-intrinsic alterations in hematopoietic stem cells (HSCs); however, the contribution of the aged microenvironment has not been adequately evaluated. Understanding the role of the bone marrow (BM) microenvironment in supporting HSC function may prove to be beneficial in treating age-related functional hematopoietic decline. Here, we determined that aging of endothelial cells (ECs), a critical component of the BM microenvironment, was sufficient to drive hematopoietic aging phenotypes in young HSCs. We used an ex vivo hematopoietic stem and progenitor cell/EC (HSPC/EC) coculture system as well as in vivo EC infusions following myelosuppressive injury in mice to demonstrate that aged ECs impair the repopulating activity of young HSCs and impart a myeloid bias. Conversely, young ECs restored the repopulating capacity of aged HSCs but were unable to reverse the intrinsic myeloid bias. Infusion of young, HSC-supportive BM ECs enhanced hematopoietic recovery following myelosuppressive injury and restored endogenous HSC function in aged mice. Coinfusion of young ECs augmented aged HSC engraftment and enhanced overall survival in lethally irradiated mice by mitigating damage to the BM vascular microenvironment. These data lay the groundwork for the exploration of EC therapies that can serve as adjuvant modalities to enhance HSC engraftment and accelerate hematopoietic recovery in the elderly population following myelosuppressive regimens.

Orthostatic Circulatory Disorders: From Nosology to Nuts and Bolts.

When patients complain of altered consciousness or discomfort in the upright posture, either relieved by recumbency or culminating in syncope, physicians may find themselves baffled. There is a wide variety of disorders that cause abnormal regulation of blood pressure and pulse rate in the upright posture. The aim of this focused review is 3-fold. First, to offer a classification (nosology) of these disorders; second, to illuminate the mechanisms that underlie them; and third, to assist the physician in the practical aspects of diagnosis of adult orthostatic hypotension, by extending clinical skills with readily available office technology.

Endothelial-specific inhibition of NF-κB enhances functional haematopoiesis.

Haematopoietic stem cells (HSCs) reside in distinct niches within the bone marrow (BM) microenvironment, comprised of endothelial cells (ECs) and tightly associated perivascular constituents that regulate haematopoiesis through the expression of paracrine factors. Here we report that the canonical NF-κB pathway in the BM vascular niche is a critical signalling axis that regulates HSC function at steady state and following myelosuppressive insult, in which inhibition of EC NF-κB promotes improved HSC function and pan-haematopoietic recovery. Mice expressing an endothelial-specific dominant negative IκBα cassette under the Tie2 promoter display a marked increase in HSC activity and self-renewal, while promoting the accelerated recovery of haematopoiesis following myelosuppression, in part through protection of the BM microenvironment following radiation and chemotherapeutic-induced insult. Moreover, transplantation of NF-κB-inhibited BM ECs enhanced haematopoietic recovery and protected mice from pancytopenia-induced death. These findings pave the way for development of niche-specific cellular approaches for the treatment of haematological disorders requiring myelosuppressive regimens.

Vascular Platform to Define Hematopoietic Stem Cell Factors and Enhance Regenerative Hematopoiesis.

Hematopoietic stem cells (HSCs) inhabit distinct microenvironments within the adult bone marrow (BM), which govern the delicate balance between HSC quiescence, self-renewal, and differentiation. Previous reports have proposed that HSCs localize to the vascular niche, comprised of endothelium and tightly associated perivascular cells. Herein, we examine the capacity of BM endothelial cells (BMECs) to support ex vivo and in vivo hematopoiesis. We demonstrate that AKT1-activated BMECs (BMEC-Akt1) have a unique transcription factor/cytokine profile that supports functional HSCs in lieu of complex serum and cytokine supplementation. Additionally, transplantation of BMEC-Akt1 cells enhanced regenerative hematopoiesis following myeloablative irradiation. These data demonstrate that BMEC-Akt1 cultures can be used as a platform for the discovery of pro-HSC factors and justify the utility of BMECs as a cellular therapy. This technical advance may lead to the development of therapies designed to decrease pancytopenias associated with myeloablative regimens used to treat a wide array of disease states.

Activation of the vascular niche supports leukemic progression and resistance to chemotherapy.

Understanding the intricate cellular components of the bone marrow microenvironment can lead to the discovery of novel extrinsic factors that are responsible for the initiation and progression of leukemic disease. We have shown that endothelial cells (ECs) provide a fertile niche that allows for the propagation of primitive and aggressive leukemic clones. Activation of the ECs by vascular endothelial growth factor (VEGF)-A provides cues that enable leukemic cells to proliferate at higher rates and also increases the adhesion of leukemia to ECs. Vascular endothelial growth factor A-activated ECs decrease the efficacy of chemotherapeutic agents to target leukemic cells. Inhibiting VEGF-dependent activation of ECs by blocking their signaling through VEGF receptor 2 increases the susceptibility of leukemic cells to chemotherapy. Therefore, the development of drugs that target the activation state of the vascular niche could prove to be an effective adjuvant therapy in combination with chemotherapeutic agents.

Endothelial Jagged-1 is necessary for homeostatic and regenerative hematopoiesis.

The bone marrow (BM) microenvironment is composed of multiple niche cells that, by producing paracrine factors, maintain and regenerate the hematopoietic stem cell (HSC) pool (Morrison and Spradling, 2008). We have previously demonstrated that endothelial cells support the proper regeneration of the hematopoietic system following myeloablation (Butler et al., 2010; Hooper et al., 2009; Kobayashi et al., 2010). Here, we demonstrate that expression of the angiocrine factor Jagged-1, supplied by the BM vascular niche, regulates homeostatic and regenerative hematopoiesis through a Notch-dependent mechanism. Conditional deletion of Jagged-1 in endothelial cells (Jag1((ECKO)) mice) results in a profound decrease in hematopoiesis and premature exhaustion of the adult HSC pool, whereas quantification and functional assays demonstrate that loss of Jagged-1 does not perturb vascular or mesenchymal compartments. Taken together, these data demonstrate that the instructive function of endothelial-specific Jagged-1 is required to support the self-renewal and regenerative capacity of HSCs in the adult BM vascular niche.

Use of different morphological techniques to analyze the cellular composition of the adult zebrafish optic tectum.

Cellular composition of the adult zebrafish (Danio rerio) optic tectal cortex was examined in this study. Morphological techniques such as 1 µm thick serial plastic sections stained with osmium tetroxide and toluidine blue, modified rapid Golgi silver impregnation, GFAP immunohistochemistry, confocal microscopy, as well as scanning and transmission electron microscopy were used. Neuronal and glial components are described and the layers of the cortex are revisited. Specific neuronal arrangements as well as unique glial/ependymal cells are described. A three dimensional rendering of the astrocytic fiber arrangement in the marginal zone is presented and a composite drawing summarizes the cellular composition of the optic tectum.

SlimPLS: a method for feature selection in gene expression-based disease classification.

A major challenge in biomedical studies in recent years has been the classification of gene expression profiles into categories, such as cases and controls. This is done by first training a classifier by using a labeled training set containing labeled samples from the two populations, and then using that classifier to predict the labels of new samples. Such predictions have recently been shown to improve the diagnosis and treatment selection practices for several diseases. This procedure is complicated, however, by the high dimensionality if the data. While microarrays can measure the levels of thousands of genes per sample, case-control microarray studies usually involve no more than several dozen samples. Standard classifiers do not work well in these situations where the number of features (gene expression levels measured in these microarrays) far exceeds the number of samples. Selecting only the features that are most relevant for discriminating between the two categories can help construct better classifiers, in terms of both accuracy and efficiency. In this work we developed a novel method for multivariate feature selection based on the Partial Least Squares algorithm. We compared the method's variants with common feature selection techniques across a large number of real case-control datasets, using several classifiers. We demonstrate the advantages of the method and the preferable combinations of classifier and feature selection technique.

Loss of chromosome 16 from renal epithelial cells in humans.

This work explores the notion that low-frequency, acquired aneuploidy may play a role in complex genetic traits such as essential hypertension. To this end, renal epithelial cells in urinary sediments and in renal cysts were examined by fluorescent in situ hybridization with DNA probes specific for the heterochromatic and centromere regions of chromosomes 16 and 1. Chromosome 16 was probed because it harbors variant genes causing monogenic hypertension. These genes have also been investigated for their role in essential hypertension. Chromosome 1 was also probed as an internal control. Higher proportions of renal epithelial cells in the urinary sediments showed monosomy of chromosome 16 than monosomy of chromosome 1 (P<0.001). We also observed in epithelial cells of renal cysts a preponderance of monosomy for chromosome 16 over monosomy for chromosome 1 (P<0.024). Low-frequency loss of heterozygosity that results from acquired monosomy of chromosome 16 and perhaps other chromosomes may contribute to expression of complex genetic traits such as essential hypertension, in which the diverse phenotypic manifestations are poorly understood.