Sinusoidal Blood Vessels Research Articles

Morphological identification and distribution comparison of telocytes in pituitary gland between normal and cryptorchid yaks

BackgroundTelocytes (TCs) is a novel type of interstitial cells in many mammals organs, which participate in the organizational metabolism, mechanical support, immunomodulation and other aspects. The aim of this study was to explore the organizational chemical characteristics of TCs in pituitary gland and their changes in cryptorchid yaks.MethodsTransmission electron microscopy (TEM), toluidine blue staining, immunofluorescence, qRT-PCR, and Western blotting may enable us to understand TCs distribution characteristics and biological functions.ResultTEM confirmed the presence of TCs in the pituitary gland with small bodies and moniliform telopodes (Tps). The Tps extending out from the cell body to the peri-sinusoidal vessels spaces, the number of Tps is closely related to the morphology of the nucleus. The most obvious changes of TCs in the pituitary gland of cryptorchid yaks is the Tps are relatively shorter and decreased secretory vesicles. H.E. and toluidine blue staining revealed that TCs not only distributed between the sinusoidal blood vessels and the glandular cell clusters, but also present on the surface of vascular endothelial cells. The co-expression of TCs biomarkers, such as Vimentin/CD34, CD117/CD34 and α-SMA/CD34, were evaluated by immunofluorescence to further determine the phenotypic characteristics of TCs. Besides, we analyzed the mRNA and protein expression of these biomarkers to determine the characteristics of TCs changes and possible biological roles. Both the mRNA and protein expression of CD117 were significantly higher in the pituitary gland of cryptorchid yaks than in the normal (p < 0.01), the protein expression of CD34 in the cryptorchid yaks was significantly higher than the normal (p < 0.01). There were no significant difference in mRNA expression of Vimentin and α-SMA (p>0.05), while the protein expression were significantly increased in the normal yaks (p < 0.05).ConclusionsIn summary, this study reports for the first time that the biological characteristics of TCs in yak pituitary gland. Although there is no significant change in the distribution characteristics, the changes in biological features of TCs in cryptorchid yaks are clear, suggesting that TCs participated in alteration in the local microenvironment of the pituitary gland. Therefore, our study provides clues for further investigating the role of TCs in the pituitary gland during the occurrence of cryptorchidism in yaks.

Skeletal stem and progenitor cells in bone physiology, ageing and disease.

Skeletal stem cells (SSCs) and related progenitors with osteogenic potential, collectively termed skeletal stem and/or progenitor cells (SSPCs), are crucial for providing osteoblasts for bone formation during homeostatic tissue turnover and fracturerepair. Besides mediating normal bone physiology, they also have important roles in various metabolic bone diseases, including osteoporosis. SSPCs are of tremendous interest because they represent prime future targets for osteoanabolic therapies and bone regenerative medicine. Remarkable progress has been made in characterizing various SSC and SSPC populations in postnatal bone. SSPCs exist in the periosteum and within the bone marrow stroma, including subsets localizing around arteriolar and sinusoidal blood vessels; they can display osteogenic, chondrogenic, adipogenic and/or fibroblastic potential, and exert critical haematopoiesis-supportive functions. However, much remains to be clarified. By the current markers, bona fide SSCs are commonly contained within broader SSPC populations characterized by considerable heterogeneity and overlap, whose common versus specific functions in health and disease have not been fully unravelled. Here, we review the present knowledge of the identity, fates and relationships of SSPC populations in the postnatal bone environment, their contributions to bone maintenance, the changes observed upon ageing, and the effect of metabolic diseases such as osteoporosis and diabetes mellitus.

Vascular biology and hemophilia

By carrying a systemic circulation, hematopoietic and vascular systems coordinately govern the functional organ connections in the body. Blood vessels play an important role in the development, regeneration, and maintenance of organs by acting as conduits for environmental factors in the blood to tissues and secreting organ-specific cytokines as angiocrine signals. Recently, it has become clear that vascular endothelial cells, which are the main constituent cells of the blood vessels and play a role in homeostasis, are diverse. It has also been established that the cells of stem cell fraction exist in endothelial cells. The vascular endothelial cells in various organs are functionally different. For example, it has been discovered that sinusoidal blood vessels in the liver produce coagulation factor VIII as an organ-specific vascular function. Determining how such tissue-/organ-specific function of the endothelial cells is induced is a topic of interest in the vascular field of study.

Novel insights into mouse models of ectopic proplatelet release

AbstractMature bone marrow (BM) megakaryocytes (MKs) produce platelets by extending proplatelets into sinusoidal blood vessels. Defects in this process can lead to thrombocytopenia and increased risk of bleeding. Mice lacking the actin-regulatory proteins Profilin 1 (PFN1), Wiskott–Aldrich Syndrome protein (WASp), Actin Related Protein 2/3 complex (Arp2/3), or adhesion and degranulation-promoting adapter protein (ADAP) display thrombocytopenia and ectopic release of (pro)platelet-like particles into the BM compartment, pointing to an important axis of actin-mediated directional proplatelet formation. The mechanism underlying ectopic release in these mice is still not completely understood. However, we hypothesized that similar functional defects account for this observation. We analyzed WASp-, ADAP-, PFN1-, and ARPC2-knockout mice to determine the role of actin reorganization and integrin activation in directional proplatelet formation. ADAP-, ARPC2-, and PFN1-deficient MKs displayed reduced adhesion to collagen, defective F-actin organization, and diminished β1-integrin activation. WASp-deficient MKs showed the strongest reduction in the adhesion assay of collagen and altered F-actin organization with reduced podosome formation. Our results indicate that ADAP, PFN1, WASp, and ARP2/3 are part of the same pathway that regulates polarization processes in MKs and directional proplatelet formation into BM sinusoids.

Targeting FAPα-expressing hepatic stellate cells overcomes resistance to antiangiogenics in colorectal cancer liver metastasis models.

Vessel co-option has been demonstrated to mediate colorectal cancer liver metastasis (CRCLM) resistance to antiangiogenic therapy. The current mechanisms underlying vessel co-option have mainly focused on “hijacker” tumor cells, whereas the function of the “hijackee” sinusoidal blood vessels has not been explored. Here, we found that the occurrence of vessel co-option in bevacizumab-resistant CRCLM xenografts was associated with increased expression of fibroblast activation protein α (FAPα) in the co-opted hepatic stellate cells (HSCs), which was dramatically attenuated in HSC-specific conditional Fap-knockout mice bearing CRCLM allografts. Mechanistically, bevacizumab treatment induced hypoxia to upregulate the expression of fibroblast growth factor–binding protein 1 (FGFBP1) in tumor cells. Gain- or loss-of-function experiments revealed that the bevacizumab-resistant tumor cell–derived FGFBP1 induced FAPα expression by enhancing the paracrine FGF2/FGFR1/ERK1/-2/EGR1 signaling pathway in HSCs. FAPα promoted CXCL5 secretion in HSCs, which activated CXCR2 to promote the epithelial-mesenchymal transition of tumor cells and the recruitment of myeloid-derived suppressor cells. These findings were further validated in tumor tissues derived from patients with CRCLM. Targeting FAPα+ HSCs effectively disrupted the co-opted sinusoidal blood vessels and overcame bevacizumab resistance. Our study highlights the role of FAPα+ HSCs in vessel co-option and provides an effective strategy to overcome the vessel co-option–mediated bevacizumab resistance.

Abstract 6154: Distinct immunosuppressive environments elicited in vessel co-opting and angiogenic colorectal cancer liver metastases and changes following treatment

Abstract Colorectal cancer (CRC) is the third most common cancer worldwide and 50% of CRC patients develop liver metastases. Colorectal cancer liver metastases (CRCLM) present as two major histological growth patterns (HGP) that predict response to treatment/survival: 1. angiogenic tumors characterized by a desmoplastic stroma separating CRC cells from the liver parenchyma and are highly angiogenic and 2. co-opting tumors where tumor cells infiltrate the parenchymal cells in the liver and grow by co-opting the sinusoidal blood vessels between the liver cell plates without sprouting angiogenesis. Angiogenic tumors receiving neoadjuvant anti-angiogenics (anti-VEGF) and chemotherapy have more than double the 5-year overall survival compared to patients with co-opting tumors who have received the same neoadjuvant regimen. In addition, our clinical data revealed that anti-angiogenics could negatively affect outcomes in patients with co-opting lesions. The goal of our study is to understand how the immune system affects the development of the two distinct CRCLM tumors. We used both Nanostring GeoMX spatial protein profiling platform, immunohistochemistry and immunofluorescence, to identify the immune landscapes in chemonaïve and treated human CRCLM tumors. We observed that angiogenic tumors were rich in both CD4 and CD8 T cells with a near absence of neutrophils and macrophages. These adaptive immune cells were also FOXP3 positive indicating an immunosuppressive phenotype. Interestingly, we observed that vessel co-opting tumors have far fewer infiltrating lymphocytes than angiogenic tumors, but interestingly have greater numbers of innate cells - mostly neutrophils and macrophages. The macrophages were further characterized as being M2, tumor promoting, leading to an immunosuppressive environment. We then went on to analyze treated tumors. For those patients treated with chemotherapy alone we observed a larger infiltration of T cells in the angiogenic tumors and the appearance of T cells infiltrating the co-opting tumors. Surprisingly, in patients treated with chemotherapy and anti-angiogenic drugs (ie. anti-VEGF), which is part of the standard of care, the co-opting tumors no longer demonstrated the presence of T cell infiltration and had a similar profile to the chemonaïve tumors. This could explain why chemotherapy in co-opting tumors had a higher overall survival compared to those treated with chemotherapy and anti-VEGF. These results suggest developing treatments aimed at reshaping the different immune landscape in angiogenic and co-opting CRCLM to yield beneficial results for patient overall survival. Furthermore, the identification of immune cells or immune mediators facilitating the development of these two tumor would identify immune cell targets for immunotherapies. Citation Format: Diane H. Kim, Thomas Z. Mayer, Stephanie K. Petrillo, Anthoula Lazaris, Peter Metrakos. Distinct immunosuppressive environments elicited in vessel co-opting and angiogenic colorectal cancer liver metastases and changes following treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6154.

Niches that regulate stem cells and hematopoiesis in adult bone marrow.

In mammals, hematopoietic stem cells (HSCs) engage in hematopoiesis throughout adult life within the bone marrow, where they produce the mature cells necessary to maintain blood cell counts and immune function. In the bone marrow and spleen, HSCs are sustained in perivascular niches (microenvironments) associated with sinusoidal blood vessels-specialized veins found only in hematopoietic tissues. Endothelial cells and perivascular leptin receptor+ stromal cells produce the known factors required to maintain HSCs and many restricted progenitors in the bone marrow. Various other cells synthesize factors that maintain other restricted progenitors or modulate HSC or niche function. Recent studies identified new markers that resolve some of the heterogeneity among stromal cells and refine the localization of restricted progenitor niches. Other recent studies identified ways in which niches regulate HSC function and hematopoiesis beyond growth factors. We summarize the current understanding of hematopoietic niches, review recent progress, and identify important unresolved questions.

A novel strategy for isolation of mice bone marrow endothelial cells (BMECs)

BackgroundIn the bone marrow microenvironment (BM), endothelial cells are individual cells that form part of the sinusoidal blood vessels called the “bone marrow endothelial-vascular niche.” They account for less than 2% of the bone marrow cells. They play essential functions by generating growth and inhibitory factors that promote the hematopoietic stem cells (HSCs) regulation. In response to inflammatory stimuli, the BMECs increase in proliferation to maintain the blood vessels’ integrity within the BM. The inflammatory response releases cytokines such as tumor necrosis factor-alpha (TNF-α) that promote vascular endothelial cells’ expansion and upregulation of adhesion molecules (ICAM-1 and VCAM-1, respectively) in the BM. However, the evaluation of mouse BMECs in the bone marrow microenvironment is scared by a lack of mouse bone marrow endothelial cell primary cultureMethodsTwo steps approach for isolation of bone marrow endothelial cells (BMECs) from mice. In brief, the bone marrow cells extracted from the mice long bones were cultured overnight with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20% fetal bovine serum (FBS) and antibiotics to separate between marrow-derived adherent and non-adherent cells. The floating cells were discarded, and the adhered section detached with accutase and BMECs selected using CD31 microbeads. The isolated BMECs were cultured in a dish pre-coated with rat-tail collagen type 1 with endothelial cells medium supplement with growth factors. The cells were verified by confocal microscopy for morphology and tube formation by matrigel assay. We validate the cells’ purity by flow cytometry, RT-qPCR, immunofluorescence staining, and immunoblotting by established BMEC markers, PECAM-1, VE-cadherin, vascular endothelial cell growth factor receptor-2 (VEGFR2), CD45, E-selectin, and endothelial selectin adhesion molecule (ESAM). Lastly, we characterize BMEC activation with recombinant TNF-α.ResultsOur method clearly defined the cells isolated have the characteristics of BMECs with the expression of CD31, VE-cadherin, E-selectin, VEGFR-2, and ESAM. The cells’ response to TNF-α indicates its inflammatory function by increasing proliferation and upregulation of adhesion molecules.ConclusionsThis study outline a simple new technique of isolating mouse BMEC primary culture and a suitable method to evaluate the function and dysregulation of BMEC in in vitro studies using mouse models.

Primary Alveolar Soft Part Sarcoma Arising in the Thyroid Gland: A Rare Case Report

Background Alveolar soft part sarcoma (ASPS) is a rare aggressive soft tissue sarcoma usually involving the soft tissue of thigh and buttock region in adults. In children, head and neck presentation is more common, with orbit and tongue being the preferred sites. Alveolar soft part sarcoma primarily arising in the thyroid gland is an extremely rare clinical entity, with only two other cases being reported in the literature to date. Case A 16-year-old boy presented with respiratory distress and enlarged thyroid gland. Hemithyroidectomy revealed well-circumscribed nests of large cells with abundant eosinophilic cytoplasm arranged in a pseudoalveolar pattern and separated by fibrous septa containing sinusoidal blood vessels. The tumor cells showed nuclear positivity for TFE3 protein and the TFE3 gene rearrangement in 79% of the nuclei. The patient's most recent chest computed tomography revealed multiple subcentimeter nodules occupying the lung bases emphasizing the aggressiveness of the disease process and the need for constant surveillance.

Liver zonation

Liver zonation

The impact of dietary tarragon (Artemisia dracunculus) on serum apelin, brain-derived neurotrophic factor, cardiac troponin concentrations and histopathology of liver tissue in laying hens housed at different stocking densities

Due to its association with several other stress factors (poultry house gases, inadequate ventilation, heat, cold and poor hygiene), the high stocking density is a major stress factor that adversely affects the health and performance of poultry and the quality of the poultry products. Therefore, this experimental study was aimed at analysing the impact of different doses of dietary tarragon (Artemisia dracunculus) on the serum apelin, plasma brain-derived neurotrophic factor (p-BDNF), and cardiac troponin I (cTnI) concentrations, and the correlation between these indicators in laying hens housed at different stocking densities. The aim of this study is to investigate the effects of adding tarragon in different ratios to laying hen rations in the 2&lt;sup&gt;nd&lt;/sup&gt; ovulation period on the cTnI, apelin, and BDNF hormone concentrations and the liver histopathology. The experiment was carried out over a period of eight weeks, with 192 Lohman Brown commercial hybrids at 50 weeks of age. Eight groups (four replicates each), composed of laying hens of equal body weight, which were housed at stocking densities of 580 cm&lt;sup&gt;2&lt;/sup&gt;/hen and 810 cm&lt;sup&gt;2&lt;/sup&gt;/hen and received 0, 1, 5 and 10 mmol/kg of tarragon (Artemisia dracunculus) in the feed, were established. At the end of the trial, 96 of the housed egg-laying hens (3 birds in each subgroup, a total of 12 birds in each group) were randomly selected and blood samples were taken from the vena subcutanea ulnaris. The samples collected were analysed for the apelin, p-BDNF, and cTnI contents. The analysis results demonstrated that tarragon supplementation had no effect on the serum apelin, p-BDNF and cTnI concentrations (P &amp;gt; 0.05). The Sub-Groups ST1, ST1.2, and ST6 presented with severe hyperaemia of the sinusoidal, portal and acinar blood vessels, whilst the hyperaemia of these blood vessels was moderate in Sub-Group ST12. Apelin, BDNF, and cTnI can act as protective factors against negative consequences of stress (e.g., stocking density or heat stress).

Live-animal imaging of native haematopoietic stem and progenitor cells.

The biology of hematopoietic stem cells (HSCs) has predominantly been studied under transplantation conditions1,2. Particularly challenging has been the study of dynamic HSC behaviors given that live animal HSC visualization in the native niche still represents an elusive goal in the field. Here, we describe a dual genetic strategy in mice that restricts reporter labeling to a subset of the most quiescent long-term HSCs (LT-HSCs) and that is compatible with current intravital imaging approaches in the calvarial bone marrow (BM)3–5. We find that this subset of LT-HSCs resides in close proximity to both sinusoidal blood vessels and the endosteal surface. In contrast, multipotent progenitor cells (MPPs) display a broader distance distribution from the endosteum and are more likely to be associated with transition zone vessels. LT-HSCs are not found in BM niches with the deepest hypoxia and instead are found in similar hypoxic environments as MPPs. In vivo time-lapse imaging reveals that LT-HSCs display limited motility at steady-state. Following activation, LT-HSCs display heterogenous responses, with some cells becoming highly motile and a fraction of HSCs expanding clonally within spatially restricted domains. These domains have defined characteristics, as HSC expansion is found almost exclusively in a subset of BM cavities exhibiting bone-remodeling activities. In contrast, cavities with low bone-resorbing activities do not harbor expanding HSCs. These findings point to a new degree of heterogeneity within the BM microenvironment, imposed by the stages of bone turnover. Overall, our approach enables direct visualization of HSC behaviors and dissection of heterogeneity in HSC niches.

A Wnt-mediated transformation of the bone marrow stromal cell identity orchestrates skeletal regeneration

Bone marrow stromal cells (BMSCs) are versatile mesenchymal cell populations underpinning the major functions of the skeleton, a majority of which adjoin sinusoidal blood vessels and express C-X-C motif chemokine ligand 12 (CXCL12). However, how these cells are activated during regeneration and facilitate osteogenesis remains largely unknown. Cell-lineage analysis using Cxcl12-creER mice reveals that quiescent Cxcl12-creER+ perisinusoidal BMSCs differentiate into cortical bone osteoblasts solely during regeneration. A combined single cell RNA-seq analysis demonstrate that these cells convert their identity into a skeletal stem cell-like state in response to injury, associated with upregulation of osteoblast-signature genes and activation of canonical Wnt signaling components along the single-cell trajectory. β-catenin deficiency in these cells indeed causes insufficiency in cortical bone regeneration. Therefore, quiescent Cxcl12-creER+ BMSCs transform into osteoblast precursor cells in a manner mediated by canonical Wnt signaling, highlighting a unique mechanism by which dormant stromal cells are enlisted for skeletal regeneration.

KIFC1 Mediates Centrosome Clustering Prior to Proplatelet Formation from Megakaryocytes

Platelets are specialized anucleate cells that circulate in the blood and serve to prevent bleeding and minimize blood vessel injury. In addition to their hemostatic functions, platelets participate in wound healing, angiogenesis, inflammation, and immunity, and are therefore central players in both maintaining normal physiology and in disease pathogenesis. Platelets are derived from their precursor cells, megakaryocytes (MKs), that reside principally in the bone marrow. During maturation, MKs undergo an altered cell cycle called endomitosis in which they replicate their DNA but avoid cell division, resulting in polyploid MKs with amplified microtubule (MT)-organizing centers called centrosomes. Subsequently, MT-dependent forces are responsible for extending long cytoplasmic protrusions called proplatelets into sinusoidal blood vessels, eventually giving rise to circulating platelets. Despite progress in elucidating key steps of platelet production, there is a conspicuous lack of understanding of what triggers mature, polyploid MKs to undergo the MT rearrangements required for proplatelet production. Using live cell imaging of mouse fetal liver-derived MKs expressing fluorescent b1-tubulin, we have identified a novel MT-based structure in MKs termed a monospindle. Our data suggest that monospindles result from polyploid MKs clustering multiple centrosomes into a centralized MT-organizing center during mitosis, leading to an enlarged array of MTs oriented towards the cell cortex. These structures were also apparent in mouse bone marrow- and human cord blood-derived MKs, suggesting that monospindle formation is a general phenomenon in MKs. Interestingly, a higher percentage of MKs contained monospindles at a timepoint directly prior to proplatelet production (50%) compared to when proplatelets were actively being produced (22%), indicating a possible role in initiating proplatelet formation. Centrosome clustering in cancer cells is mediated by the MT-based mitotic motor protein, KIFC1. Consistently, we found that small molecule inhibition of KIFC1 decreased the percentage of MKs containing monospindles (55% ctrl vs. 6% KIFC1 inhibitor). Strikingly, KIFC1 inhibitor treatment also drastically reduced the percentage of MKs producing proplatelets (peak proplatelet formation: 40% ctrl vs. 5% KIFC1 inhibitor), suggesting that KIFC1-mediated centrosome clustering into monospindles is important for proplatelet production. To test how KIFC1 contributes to these phenotypes, we assessed its expression at different timepoints by Western blot and detected increased KIFC1 levels in more mature MKs preceding proplatelet formation. Cell sorting of MKs into distinct ploidy populations followed by Western blot showed that KIFC1 expression increased with higher ploidy. Thus, our results lead us to suggest a working model in which elevated KIFC1 levels in mature MKs drive monospindle formation to trigger proplatelet formation. Investigating the role of KIFC1-mediated monospindle formation for initiating proplatelet formation could yield a coherent, molecular understanding of how mature, polyploid MKs reorganize MTs for proplatelet production. In addition, these data will help inform basic cell biology, as there are important parallels between centrosome clustering in MKs and cancer cells. Finally, our findings could yield novel therapeutic strategies for treating patients with thrombocytopenia (low platelet counts) by directly stimulating platelet production from mature MKs residing in the bone marrow. Disclosures Italiano: Platelet Biogenesis: Employment, Equity Ownership; Ionis Research Funding: Research Funding.

1005 - THE IDENTIFICATION OF A PERI-ARTERIOLAR NICHE FOR LYMPHOID PROGENITORS AND OSTEOGENIC PROGENITORS IN THE BONE MARROW

Leptin Receptor-expressing (LepR+) stromal cells in adult bone marrow are highly enriched for skeletal stem cells and are the main source of osteoblasts and adipocytes in addition to SCF and Cxcl12 for HSC maintenance (Nature 481:457; Nature 495:231; Cell Stem Cell 15:154). We discovered that a subset of these cells produce a previously uncharacterized bone-forming growth factor, Clec11a/Osteolectin, which promotes the osteogenic differentiation of LepR+ cells and is required for the maintenance of adult skeletal bone mass (eLife 5:e18782). Osteolectin promotes osteogenic differentiation by binding to a11b1 integrin and promoting Wnt pathway activation (eLife 8:e42274). To test if Osteolectin expression could help resolve some of the heterogeneity among LepR+ cells in vivo, we generated a knock-in tdTomato reporter. We found that Osteolectin was exclusively expressed by peri-arteriolar LepR+ cells. The Osteolectin+ cells expressed osteogenic genes while the Osteolectin− cells expressed adipogenic genes. Fate mapping showed that the Osteolectin+ cells gave rise to osteoblasts but not adipocytes in vivo. Conditional deletion of Scf in Osteolectin+ cells did not affect the frequency or function of hematopoietic stem cells (HSCs) or most restricted hematopoietic progenitors in the bone marrow, consistent with our observation that most HSCs and restricted progenitors are associated with sinusoidal blood vessels (Nature 526:126; Cell Stem Cell 24:477); however, common lymphoid progenitors were depleted. This suggests the existence of a peri-arteriolar niche for a subset of lymphoid progenitors in the bone marrow. HSCs are maintained primarily in peri-sinusoidal niches while lymphoid progenitors are maintained in peri-arteriolar and endosteal niches.

Abstract 5151: Deciphering the molecular mechanisms driving infiltrative histopathological type of colorectal cancer liver metastases

Abstract Colorectal carcinoma (CRC) remains the second leading cause of cancer death in the western world. Over 50% of CRC patients develop liver metastases (LM) and 90% will succumb to their disease. Liver resection of the LMs provides the only possibility of cure, but only 20% of colorectal cancer liver metastases (CRCLM) patients are resectable. The combination of angiogenic inhibitors (AI: anti-VEGF) with chemotherapy is the current form of treatment. Unfortunately, 65-70% of the patients continue on chemotherapy until resistance develops and then are treated with second, third and some times a fourth line of treatment, with an expected median overall survival of 24-28 months. We have no way of identifying those CRCLM patients that would respond/benefit to the addition of anti-angiogenic therapies (e.g. Bevacizumab). Recently we have identified two CRCLM histologic growth patterns (HGP) that predict treatment response and survival: 1) Desmoplastic (DHGP), a desmoplastic ring separating cancer cells from the liver parenchyma and lesions grow by angiogenesis; 2) Replacement or infiltrative (RHGP), tumor cells infiltrate the parenchymal cells in the liver as the lesions grow by co-opting the sinusoidal blood vessels between the liver cell plates. We showed that CRCLM patients with predominantly desmoplastic HGP metastasis receiving AIs plus chemotherapy have more than double the 5-year overall survival compared to patients with replacement HGP who have received the same treatment. In addition, our clinical data revealed that Angiogenic Inhibitors could negatively affect outcomes in patients with replacement HGPs. These non-angiogenic lesions do not respond to angiogenic inhibitors. To further our understanding of the molecular differences between the two HGPs we demonstrated by knocking out ARPC3 (Actin-related protein 2/3 complex subunit 3, involved in actin polymerization) in the human colon cancer cell line, HT-29, that cancer cell motility is a crucial process that regulates histological growth pattern in CRCLM. HT29 CRC cells injected directly into the mouse liver grow into replacement HGPs, while HT29s silenced for ARPC3 grow into desmoplastic HGPs lesions. However, the molecular mechanisms that regulate ARPC3 in CRCLM remain unknown. To further dissect the molecular mechanisms differentiating desmoplastic from replacement HGPs, we performed RNA-seq analysis of CRCLM lesions from chemonaïve patients. Our data revealed that both TGFβ1 and RUNX1 were upregulated in RHGP comparing to DHGP lesions. This has been further validated by immunoblotting and immunohistochemistry. Consistently, RUNX1 has been reported as a downstream of TGFβ1 and transcriptional factor for ARPC3. Collectively, our data suggests that TGFβ1 and RUNX1 contribute to the formation of infiltrative type of colorectal cancer liver metastases possibly through upregulation of ARPC3. Note: This abstract was not presented at the meeting. Citation Format: Miran Rada, Anthoula Lazaris, Stephanie Petrillo, Abdellatif Amri, Peter Metrakos. Deciphering the molecular mechanisms driving infiltrative histopathological type of colorectal cancer liver metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5151.

Recent advances in mechanisms of megakaryopoiesis and platelet formation

Platelets are specialized cells, which are functioned to prevent bleeding and minimize blood vessel injury, and are also the predominating factor in the processes of hemostasis and thrombosis, while implicate in other processes including inflammation, innate immunity, angiogenesis and tumor metastasis. Among current mechanisms of platelet formation, platelets are produced by megakaryocytes, which are themselves generated by a process of controlled differentiation and maturation of bone marrow hematopoietic stem cells (HSC) and progenitor cells. Megakaryocytes release platelets by extending long, branching proplatelets, into sinusoidal blood vessels. Bone marrow cavity and extracellular matrix composition together with cytokines as thrombopoietin (TPO) are key regulators of megakaryopoiesis by supporting cell differentiation and platelet formation. Understanding the mechanisms of megakaryocyte differentiation and platelet formation can provide a solid theoretical basis for the treatment of platelet-related diseases. This review summarizes the current scientific research progress in the mechanisms of megakaryopoiesis and thrombopoiesis. Key words: Megakaryocytes; Cell differentiation; Thrombopoiesis; Transcription factors; Apoptosis; Megakaryopoiesis

Flavonoid genistein protects bone marrow sinusoidal blood vessels from damage by methotrexate therapy in rats.

Cancer chemotherapy can cause significant damage to the bone marrow (BM) microvascular (sinusoidal) system. Investigations must now address whether and how BM sinusoidal endothelial cells (SECs) can be protected during chemotherapy. Herein we examined the potential protective effects of genistein, a soy-derived flavonoid, against BM sinusoidal damage caused by treatment with methotrexate (MTX). The groups of young adult rats were gavaged daily with genistein (20 mg/kg) or placebo. After 1 week, rats also received daily injections of MTX (0.75 mg/kg) or saline for 5 days and were killed after a further 4 days. Histological analyses showed that BM sinusoids were markedly dilated ( p < 0.001) in the MTX-alone group but were unaffected or less dilated in the genistein+MTX group. In control rats, genistein significantly enhanced expression of vascular endothelial growth factor (VEGF; p < 0.01), particularly in osteoblasts, and angiogenesis marker CD31 ( p < 0.001) in bone. In MTX-treated rats, genistein suppressed MTX-induced apoptosis of BM SECs ( p < 0.001 vs MTX alone group) and tended to increase expression of CD31 and VEGF ( p < 0.05). Our in vitro studies showed that genistein in certain concentrations protected cultured SECs from MTX cytotoxic effects. Genistein enhanced tube formation of cultured SECs, which is associated with its ability to induce expression of endothelial nitric oxide synthase and production of nitric oxide. These data suggest that genistein can protect BM sinusoids during MTX therapy, which is associated, at least partially, with its indirect effect of promoting VEGF expression in osteoblasts and its direct effect of enhancing nitric oxide production in SECs.

ADAP deficiency impairs megakaryocyte polarization with ectopic proplatelet release and causes microthrombocytopenia

AbstractBone marrow (BM) megakaryocytes (MKs) produce platelets by extending proplatelets into sinusoidal blood vessels. Defects in thrombopoiesis can lead to thrombocytopenia associated with increased bleeding tendency. Recently, the platelet disorder congenital autosomal-recessive small-platelet thrombocytopenia (CARST) was described; it is caused by mutations in the adhesion and degranulation-promoting adaptor protein (ADAP; synonym: FYB, SLAP130/120) gene, and characterized by microthrombocytopenia and bleeding symptoms. In this study, we used constitutive ADAP-deficient mice (Adap−/−) as a model to investigate mechanisms underlying the microthrombocytopenia in CARST. We show that Adap−/− mice display several characteristics of human CARST, with moderate thrombocytopenia and smaller-sized platelets. Adap−/− platelets had a shorter life span than control platelets, and macrophage depletion, but not splenectomy, increased platelet counts in mutant mice to control levels. Whole-sternum 3-dimensional confocal imaging and intravital 2-photon microscopy revealed altered morphology of ADAP-deficient MKs with signs of fragmentation and ectopic release of (pro)platelet-like particles into the BM compartment. In addition, cultured BM-derived MKs lacking ADAP showed reduced spreading on extracellular matrix proteins as well as activation of β1 integrins, impaired podosome formation, and displayed defective polarization of the demarcation membrane system in vitro. MK-/platelet-specific ADAP-deficient mice (PF4-cre) also produced fewer and smaller-sized platelets and released platelets ectopically. These data demonstrate that the abnormal platelet production in the mutant mice is an MK-intrinsic defect. Taken together, these results point to an as-yet-unidentified role of ADAP in the process of MK polarization and platelet biogenesis.

Pediatric cranial intraosseous hemangiomas: a review.

Hemangiomas are benign, slow-growing tumors composed of sinusoidal blood vessels. Skeletal hemangiomas are uncommon and are mostly vertebral, followed by cranial in location. Cranial hemangiomas are very rarely encountered in children. Authors report a 12-year girl who presented with a painless enlarging mass over the parietal scalp for 3months. Imaging revealed a left parietal intraosseous lytic mass with a sunburst appearance. Enbloc removal and cranioplasty was performed, and histopathology was suggestive of hemangioma. We reviewed the literature on pediatric cranial intraosseous hemangiomas (PCIH) (age ≤18years) by searching online database. Including ours, a total of 24 cases were analyzed. Mean age was 10.2years (range 4months-17years). Eight were in the first decade and 16 were in the second decade. Male:female ratio was 12:12 (1:1). A painless palpable mass was the commonest presenting feature. Parietal and frontal bones were most commonly involved. Intracranial extension was noted in cases. Mean size of the lesion was 5cm (range 1-12cm). Twenty-two underwent primary surgical removal while two had additional pre-operative embolization. Surgical procedures were craniectomy alone (n-3), craniectomy+cranioplasty (n-6), tumor excision+remodeling (n-3), and tumor debulking (n-2). Histopathology was cavernous type in majority of cases. Mean follow-up duration was 11.8months (range 2-38months). There were no recurrences. One patient died due to systemic infection. Ours is the first review exclusively on PCIH. Although rare, they need to be considered in the differential diagnosis of lytic skull lesions in children. Enbloc removal with cranioplasty is the preferred treatment in vault hemangiomas, while embolization followed by debulking would suffice in large cranial base lesions.