OBJECTIVE

Radiotherapy is one of the important treatment modalities for tumors of pelvic organs. The fixed location of the rectum and its anatomic relationship with other pelvic organs makes it prone to radiation injury resulting in chronic radiation proctopathy in 5% to 20% of patients. Endothelial dysfunction has been associated with a number of pathophysiological processes. Endothelial cells synthesize and release various factors that regulate angiogenesis, inflammatory responses, hemostasis, as well as vascular tone and permeability.

METHODS

Rectum tissue samples from 20 patients with established chronic radiation proctopathy were analysed for the expression of genes related to oxidative stress, tissue hypoxia, angiogenesis, and inflammation [endoglin (ENG), activin receptor-like kinase 1 (ALK1), platelet endothelial cell adhesion molecule 1 (PECAM), vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF2), hypoxia-inducible factor 1 (HIF-1), and interleukin-1 beta (IL-1β)].

RESULTS

Overexpression of HIF-1, VEGF, FGF2, and IL-1β was detected in affected tissue. For the first time, a significant suppression of activin receptor-like kinase 1 and ENG could be revealed.

CONCLUSIONS

The data provided here allow further insight into the pathogenesis of radiation-induced rectum injury. Radiation-induced damage is not confined to a single event but involves complex signaling between different pathways, enhancing and maintaining the processes that lead to mucosal damage. The results indicate that postradiation tissue hypoxia is critical for fibrosis, which involves changes in the expression of profibrotic and angiogenic factors in rectal tissue.

A 17-year-old female presented with axillary lymphoadenopathy, which, on biopsy, demonstrated an anaplastic large cell lymphoma of the lymphohistiocytic type (ALCL-LH). The tumor cells expressed the CD30 antigen and reacted with the ALK1 antibody, suggesting the presence of the nucleophosmin-anaplastic large cell lymphoma kinase (NPM/ALK) fusion protein. No other adenopathy was found. Following a wide excision of the lymph node and without postoperative treatment, the patient remains free of disease 5 years later. This case demonstrates the potential curability of patients with early stages of ALCL by local treatment.

T-cell lymphoma in patients infected with HIV is much less common than B-cell lymphoma. We describe two cases of HIV-associated extranodal lymphoma that showed Toutonlike tumor giant cells and mononuclear large lymphoma cells. Both cell types expressed T-cell-associated antigens, including CD3, CD5, CD43, and CD45RO, and were CD4- and CD30-positive and negative for all B-lineage-associated antigens. Both cases showed T-cell receptor gamma chain gene rearrangements using the polymerase chain reaction and were negative for the Epstein-Barr virus by in situ hybridization. Despite the expression of CD30 by the multinucleated cells, both cases were negative for ALK1 by immunohistochemistry and failed to show evidence of the nucleophosmin-anaplastic lymphoma kinase fusion product characteristic of t(2;5) using the reverse-transcriptase polymerase chain reaction. Although rare, CD4-positive, T-cell lymphoma with Toutonlike giant cells may be a distinct type of HIV-associated malignant lymphoma.

To determine the efficacy of dalantercept, a soluble ALK1 inhibitor receptor fusion protein, in patients with persistent or recurrent ovarian carcinoma and related malignancies.

Eligibility criteria included measurable disease, 1-2 prior cytotoxic regimens and GOG performance status (PS) ≤2. Dalantercept was administered subcutaneously at 1.2 mg/kg every 3 weeks until disease progression or development of unacceptable toxicity. The primary null hypothesis was the probability of response ≤0.10 and the probability of 6-month progression-free survival without receipt of non-protocol therapy (event-free survival at 6 months, EFS6) ≤0.15, using RECIST 1.1 criteria.

The first stage was closed after enrollment of 30 participants with median age of 56.5 years, high-grade serous histology in 76.7%, 2 prior regimens in 46.7%, and platinum-free interval <6 months in 73.3%. All participants discontinued dalantercept, 24 (80.0%), 5 (16.7%) and 1 (3.3%) due to progression, toxicity, and other reason, respectively. The median number of treatment cycles per patient was 2 (range 1-29). There were six treatment-related grade 3 AEs and no grade ≥4 AEs. There were no objective responses. EFS6 was reached in 20% (6 out of 30 participants, 90% CI 9.1% to 35.7%).

Though safe, dalantercept as administered had limited efficacy in this patient population overall.

Candida maltosa can assimilate n-alkane as a sole carbon source and cytochromes P450ALK (P450ALK) are critical for the first oxidation step. Four major P450ALKs that are encoded by genes ALK1, ALK2, ALK3 and ALK5 are induced by n-alkane and repressed by glucose at the transcriptional level. In the present work, we found that all these four genes but ALK5 are transcriptionally activated in response to a peroxisome proliferator, clofibrate. This is the first report on the peroxisome proliferator responsive gene expression in lower eucaryotes.

Disruption of an SCS2 ortholog impaired the growth of the alkane-assimilating yeast Yarrowia lipolytica on n-alkanes, particularly on n-decane, although the mRNA level of the ALK1 gene encoding a highly inducible cytochrome P450ALK was not much affected. The same disruption did not cause inositol auxotrophy, implying that Y. lipolytica SCS2 has a different function from its Saccharomyces cerevisiae counterpart.

Background & aims: Intestinal epithelial cell (IEC) barrier dysfunction is critical to the development of Crohn's disease (CD). However, the mechanism is understudied. We recently reported increased microRNA-31-5p (miR-31-5p) expression in colonic IECs of CD patients, but downstream targets and functional consequences are unknown.

Methods: microRNA-31-5p target genes were identified by integrative analysis of RNA- and small RNA-sequencing data from colonic mucosa and confirmed by quantitative polymerase chain reaction in colonic IECs. Functional characterization of activin receptor-like kinase 1 (ACVRL1 or ALK1) in IECs was performed ex vivo using 2-dimensional cultured human primary colonic IECs. The impact of altered colonic ALK1 signaling in CD for the risk of surgery and endoscopic relapse was evaluated by a multivariate regression analysis and a Kaplan-Meier estimator.

Results: ALK1 was identified as a target of miR-31-5p in colonic IECs of CD patients and confirmed using a 3'-untranslated region reporter assay. Activation of ALK1 restricted the proliferation of colonic IECs in a 5-ethynyl-2-deoxyuridine proliferation assay and down-regulated the expression of stemness-related genes. Activated ALK1 signaling increased colonic IEC differentiation toward colonocytes. Down-regulated ALK1 signaling was associated with increased stemness and decreased colonocyte-specific marker expression in colonic IECs of CD patients compared with healthy controls. Activation of ALK1 enhanced epithelial barrier integrity in a transepithelial electrical resistance permeability assay. Lower colonic ALK1 expression was identified as an independent risk factor for surgery and was associated with a higher risk of endoscopic relapse in CD patients.

Conclusions: Decreased colonic ALK1 disrupted colonic IEC barrier integrity and was associated with poor clinical outcomes in CD patients.

Keywords: ALK1; Inflammatory Bowel Disease; Intestinal Epithelial Barrier; miR-31.

Bone morphogenetic protein (BMP)-9 and BMP10 are circulating ligands that mediate endothelial cell (EC) protection via complexes of the type I receptor, ALK1, and the type II receptors, the activin type-IIA and bone morphogenetic type II receptors. We previously demonstrated that BMP9 induces the expression of interleukin-6, interleukin-8 and E-selectin in ECs and may influence their interactions with monocytes and neutrophils. We asked whether BMP9 and BMP10 regulate the expression of Chemokine (C-C motif) ligand 2 (CCL2), a key chemokine involved in monocyte-macrophage chemoattraction. Here, we show that BMP9 and BMP10 repress basal CCL2 expression and release from human pulmonary artery ECs and aortic ECs. This was dependent on ALK1 and co-dependent on ACTR-IIA and BMPR-II. Assessment of canonical Smad signalling indicated a reliance of this response on Smad4. Of note, Smad1/5 signalling contributed only at BMP9 concentrations similar to those in the circulation. In the context of inflammation, BMP9 did not alter the induction of CCL2 by TNF-α. As CCL2 promotes monocyte/macrophage chemotaxis and endothelial permeability, these data support the concept that BMP9 preserves basal endothelial integrity.

Keywords: BMP10; BMP9; CCL2; Endothelial; TNF.

The selective progesterone receptor modulator mifepristone (MF) may act as a potent antiproliferative agent in different steroid-dependent cancers due to its strong antagonistic effect on the nuclear progesterone receptor (PGR). Hereby, we analyzed the effects of MF treatment on Leydig cell tumor (LCT) progression in a transgenic mouse model (inhibin-α promoter-driven SV40 T-antigen), as well as on LCT (BLTK-1 and mLTC-1) cell proliferation. MF significantly stimulated the proliferation of LCT in vitro. Similarly, a 1-mo MF or P4 treatment stimulated LCT tumor growth in vivo. Traceable/absent classical Pgr or nonclassical membrane PRs α, β, γ and Pgrmc2, but abundant membrane Pgrmc1 expression, was found in LCTs. MF did not activate glucocorticoid or androgen receptors in LCTs. Functional analysis showed that PGRMC1 is required for MF and P4 to stimulate the proliferation and invasiveness of LCTs. Accordingly, MF and P4 induced PGRMC1 translocation into the nucleus and thereby stimulated the release of TGFβ1 in LCT cells. MF and P4 treatments upregulated Tgfbr1, Tgfbr2, and Alk1 expression and stimulated TGFβ1 release in LCT cells. Our findings provide novel mechanistic insights into the action of MF as a membrane PR agonist that promotes LCT growth through PGRMC1 and the alternative TGFβ1 signaling pathway.

Keywords: PGRMC1; TGFβ; leydig cell tumor; mifepristone; progesterone; progesterone receptors.

Anaplastic large cell lymphoma (ALCL) possesses a broad morphological spectrum. Currently, we present a case of ALK-positive ALCL presenting with an alveolar growth pattern in a 22-year-old Chinese female. This patient complained of a progressively enlarged mass in the right axillary region for 6 months. Excisional biopsy revealed a well-developed alveolar structure with nests of dyscohesive tumor cells separated by delicate fibrovascular septae. The large pleomorphic cells have irregular nuclei with prominent nucleoli and fine chromatin and abundant pale cytoplasm. The neoplasm stained positively for CD2, CD3ε, CD30, ALK1, EMA and cytotoxic molecules (TIA1 and Granzyme B). Cytogenetic study via interphase Fluorescence in-Situ Hybridization disclosed the rearrangement involving ALK gene. The patient received 6 cycles of CHOP chemotherapy and achieved complete remission. She is alive in good condition up to the present. Our case is biologically similar to the conventional ALK-positive ALCLs and may just represent an unusual morphological appearance.

We reviewed 18 cases in which morphology was intermediate between Hodgkin's disease (HD) and anaplastic large cell lymphoma (ALCL). Eight cases exhibited the usual CD30+, CD15+/-, null cell phenotype of classic HD but were rich in neoplastic cells with sinusoidal infiltrating pattern. In this group, there was no expression of antigens (EMA, BNH9, CBF78) associated with ALCL, and only two were positive for Epstein-Barr virus (EBV). Ten EBV negative cases fit the description of HD like ALCL by variable expression of antigens unassociated with HD. EMA was clearly and strongly expressed in all ten, whereas antigens recognized by BNH9 and CBF78 were expressed in four and three cases, respectively. Focal expression of CD45 and CD43 was observed in half of these cases. In only one case was the t(2.5) translocation detected with the new monoclonal antibody, ALK1. Therefore, the expression of EMA, BNH9 and CBF78, often in concert without CD15 and without the specific translocation, appears currently to be the most probable phenotype and genotype of HD like ALCL. There was a tendency for aggressive behaviour of the disease considered HD like ALCL. Whether such patients will benefit from a therapeutic strategy that takes into account both phenotype and genotype remains to be discovered.

A novel set of pyrrolizine-5-carboxamides has been synthesized and evaluated for their anticancer potential against human breast MCF-7, lung carcinoma A549 and hepatoma Hep3B cancer cell lines. Compound 10c was the most active against MCF-7 with IC50 value of 4.72µM, while compound 12b was the most active against A549 and Hep3B cell lines. Moreover, kinases/COXs inhibition and apoptosis induction were suggested as potential molecular mechanisms for the anticancer activity of the novel pyrrolizines based on their structural features. The new compounds significantly inhibited COX-1 and COX-2 with IC50 values in the ranges of 5.78-11.96µM and 0.1-0.78µM, respectively with high COX-2 selectivity over COX-1. Interestingly, the most potent compound in MTT assay, compound 12b, exhibited high inhibitory activity against COX-2 with selectivity index (COX-1/COX-2)>100. Meanwhile, compound 12b displayed weak to moderate inhibition of six kinases with inhibition% (7-20%) compared to imatinib (inhibition%=1-38%). The results of cell cycle analysis, annexin V PI/FITC apoptosis assay and caspase-3/7 assay revealed that compound 12b has the ability to induce apoptosis. The docking results of compound 12b into the active sites of COXs, ALK1 and Aurora kinases indicated that it fits nicely inside their active sites. Overall, the current study highlighted the significant anticancer activity of the newly synthesized pyrrolizines with a potential multi-targeted mechanism which could serve as a base for future studies and further structural optimization into potential anticancer agents.

OBJECTIVE

To investigate possible mechanism of toxicarioside A in HS-5 bone stromal cells.

METHODS

HS-5 bone stromal cells were cultured in media supplemented with various concentrations of toxicarioside A or control DMSO (not treatment). Endoglin and TGF-β were detected by Northern and Western blot analysis and quantified in a standard method. Downstream molecules of endoglin and TGF-β (Smad1, Smad2 and their active phosphorylated counterparts, pSmad1 and pSmad2) were also detected and quantified by Western blot analysis. In addition, cell proliferation assay and small interfering RNA (siRNA) against endoglin were used to certificate the function of endolgin in the HS-5 cells.

RESULTS

Compared with the not treated (0 μg/mL) or DMSO treated control HS-5 cells, HS-5 cells treated with toxicarioside A were found significant attenuation of endolgin and TGF-β expression. Significant inhibition of cell proliferation was also found in the HS-5 cells treated with toxicarioside A. ALK1-related Smad1 and ALK5-related Smad2 were decreased in HS-5 cells treated with toxicarioside A. In addition, phosphorylated Smad1 (pSmad1) and Smad2 (pSmad2) were also found attenuation in toxicarioside A-treated HS-5 cells. RNA interference showed that blockage of endoglin by siRNA also decreased Smad1 and Smad2 expression in HS-5 cells.

CONCLUSIONS

Our results indicate that toxicarioside A can influence bone marrow stromal HS-5's function and inhibit HS-5 cell proliferation by alteration of endoglin-related ALK1 (Smad1) and ALK5 (Smad2) signaling.

There have been recent reports of a rare variant of renal cell carcinoma associated with upregulation of the anaplastic lymphoma kinase gene (ALK) arising as a consequence of chromosomal translocations. The tumours were described as having a characteristic morphology. Here, we describe a case with similar morphology characterised by eosinophilic cells, abundant intracytoplasmic lumina and scattered large ganglion-like tumour cells. There was focal staining for ALK demonstrated by immunohistochemistry. However, rather than exhibiting a chromosomal translocation involving ALK, the use of FISH and a break-apart probe demonstrated that there was increased copy number of intact 2p23, the chromosomal region containing the ALK gene. Furthermore, the use of comparative genomic hybridisation showed increase of the whole of chromosome 2 along with chromosomes 6 and 17. There was no evidence of loss of 3p nor of trisomy of 7 associated with clear cell and papillary carcinoma, respectively. We suggest that this demonstrates a novel mechanism of upregulation of ALK activity by increased copy number occurring during the development of a renal carcinoma with the characteristic ALK-associated morphology.

Bone morphogenetic protein 10 (BMP10) is a cardiac peptide growth factor, belonging to the transforming growth factor beta (TGFβ) superfamily, that critically controls cardiovascular development, growth, and maturation. It has been shown that BMP10 elicits its intracellular signaling through a receptor complex of activin receptor-like kinase 1 (ALK1) with morphogenetic protein receptor type II (BMPR2A) or activin receptor type-2A (ActR2A). Previously, we generated and characterized a transgenic mouse line expressing BMP10 from the α-myosin heavy chain gene promoter (αMHC-Bmp10), and found that these mice have normal cardiac hypertrophic responses to both physiological and pathological stimuli. In this study, we report that these transgenic mice exhibit significantly reduced levels of cardiomyocyte apoptosis and cardiac fibrosis in response to a prolonged administration of the β-adrenoceptor agonist isoproterenol (ISO). We further confirmed this cardioprotective function with a newly generated conditional Bmp10 transgenic mouse line, in which Bmp10 was activated in adult hearts by tamoxifen. Moreover, the intraperitoneal administration of recombinant human BMP10 (rhBMP10) was found to effectively protect hearts from injury, suggesting potential therapeutic utility of using BMP10 to prevent heart failure. Gene profiling and biochemical analyses indicated that BMP10 activates the SMAD-mediated canonical pathway and, unexpectedly, also the signal transducer and activator of transcription 3 (STAT3)-mediated signaling pathway both in vivo and in vitro. Additional findings further supported the notion that BMP10's cardioprotective function likely is due to its dual activation of SMAD- and STAT3-regulated signaling pathways, promoting cardiomyocyte survival and suppressing cardiac fibrosis.

OBJECTIVE

To analyze the clinical features and pathogenic gene of the patients with hereditary hemorrhagic telangiectasia (HHT).

METHODS

The clinical features of 3 HHT families were collected. And the patients were diagnosed according to clinical diagnostic analyzed criteria of HHT, the ACVRL1 gene screened and the conservation of mutation protein.

RESULTS

Three probands and 1 patient were diagnostic for HHT and 2 patients were suspected. In family I, there was a missense mutation of ACVRL1 gene in c.287A>> G on 2 patients, leading to the transferal of amino acids from Asn to Ser at 96(th) place. In family II, there was a missense mutation of c.1271C>> T on ACVRL1 in 2 patients, leading to the transfer of amino acids from Pro to Leu at 424(th) place. In family III, there was a deletion mutation of c.147delC on ACVRL1 so as to produce only the former 53 amino acids of ALK1 protein. Through an analysis of multi-species conservation, the mutations were conserved between multiple species. By querying the National Center for Biotechnology Information (NCBI) database, we confirmed that the mutation was not of a single nucleotide polymorphism (SNP).

CONCLUSIONS

The genetic screening of HHT patients may identify their virulence gene. And genetic screening of their offspring is helpful for the early diagnosis and prevention before disease onset.

Rationale: Brain arteriovenous malformations (AVMs) are abnormal tangles of vessels where arteries and veins directly connect without intervening capillary nets, increasing the risk of intracerebral hemorrhage and stroke. Current treatments are highly invasive and often not feasible. Thus, effective non-invasive treatments are needed. We previously showed that AVM brain endothelial cells (AVM-BEC) secreted higher vascular endothelial growth factor (VEGF) and lower thrombospondin-1 (TSP-1) levels than control BEC; and that miR-18a normalized AVM-BEC function and phenotype, although its mechanism remained unclear. Objective: To elucidate the mechanism of action and potential clinical application of miR-18a as an effective non-invasive treatment to selectively restore the phenotype and functionality of AVM vasculature. Methods and Results: The molecular pathways affected by miR-18a in patient-derived BECs and AVM-BECs were determined by western-blot, RT-qPCR, ELISA, co-IP, immunostaining, knockdown and overexpression studies, flow cytometry, and luciferase reporter assays. MiR-18a was shown to increase TSP-1 and decrease VEGF by reducing plasminogen activator inhibitor (PAI-1/SERPINE1) levels. Furthermore, miR-18a decreased the expression of bone morphogenetic protein 4 (BMP4) and hypoxia inducible factor 1α (HIF-1α), blocking the BMP4/activin-like kinase 2 (ALK2)/ALK1/ALK5 and Notch signaling pathways. As determined by Boyden chamber assays, miR-18a also reduced the abnormal AVM-BEC invasiveness, which correlated with a decrease in MMP2, MMP9 and ADAM10 levels. In vivo pharmacokinetic studies showed that miR-18a reaches the brain following intravenous (IV) and intranasal (IN) administration. IN co-delivery of miR-18a and NEO100, a good manufacturing practices (GMP)-quality form of perillyl alcohol (POH), improved the pharmacokinetic profile of miR-18a in the brain without affecting its pharmacologic properties. Ultra-high-resolution computed tomography angiography and immunostaining studies in an Mgp-/- AVM mouse model showed that miR-18a decreased abnormal cerebral vasculature, and restored the functionality of the bone marrow, lungs, spleen and liver. Conclusions: MiR-18a may have significant clinical value in preventing, reducing and potentially reversing AVM.

Keywords: BMP4; HIF-1α; arteriovenous malformation (AVM); endothelial cells (EC); miR-18a.

Anaplastic lymphoma kinase-positive large B-cell lymphoma (ALK+ LBCL) is a rare distinct type of non-Hodgkin's lymphoma that arises in association with alterations of the ALK gene. This distinct disease entity is typically associated with an aggressive clinical course and appears in light microscopic preparations as a monomorphic population of large, immunoblast-like cells. In this report, we describe a case of ALK+ LBCL diagnosed by transgastric endoscopic ultrasound-guided fine needle aspiration (EUS FNA) of splenic hilar lymph nodes. Modified Giemsa stained direct smears from the FNA sample demonstrated large lesional cells with foamy cytoplasm and macronucleoli admixed with small lymphocytes in tigroid backgrounds, mimicking the cytologic appearance of seminoma. Ancillary immunohistochemical studies subsequently confirmed the diagnosis of ALK+ LBCL with the lesional cells being immunoreactive for CD138, VS38c, MUM1, ALK1, and lambda light chain. The cohesiveness of the cells, the cellular morphology, and the tigroid backgrounds were all pitfalls for accurate diagnosis of this rare specific type of lymphoid malignancy by cytology. To our knowledge this is the first case report detailing the diagnosis of ALK+ LBCL by EUS FNA and the first report describing a glycogen-rich tigroid background in direct FNA smears. Establishing a refined diagnosis in cases of this rare form of LBCL is necessary, as therapies targeting ALK may be of value in clinical management. Diagn. Cytopathol. 2017;45:148-155. © 2016 Wiley Periodicals, Inc.

A potential role of haptoglobin in arterial restructuring has been suggested, and our previous study demonstrated that prohaptoglobin, the precursor of haptoglobin, stimulates endothelial angiogenesis. However, the mechanisms underlying the angiogenic effects of prohaptoglobin are still unclear. Here, we investigated angiogenic signaling induced by prohaptoglobin using human umbilical vein endothelial cells. Prohaptoglobin upregulated the expression of placental growth factor (PlGF), vascular endothelial growth factor (VEGF)-A, and VEGF receptor 1 and 2, and also induced cell migration and tube network formation. PlGF knockdown attenuated these angiogenic effects of prohaptoglobin. Furthermore, a transcription factor profiling assay indicated that Smad is involved in PlGF expression in response to prohaptoglobin. Transforming growth factor-β1 (TGF-β1) expression and Smad1/5 phosphorylation were also induced by prohaptoglobin treatment. Blockade of TGF-β1 signaling using the TGF-β receptor kinase inhibitor LY2109761 or Smad1/5 siRNA reduced the prohaptoglobin-induced PlGF expression and in vitro tube formation. Knockdown of the TGF-β receptor ALK1, but not ALK5, with a specific siRNA blocked the Smad1/5 phosphorylation and PlGF expression induced by prohaptoglobin. These findings suggest that the angiogenic effects of prohaptoglobin are dependent on PlGF and mediated via a TGF-β1-ALK1-Smad1/5-PlGF/VEGFR1-VEGF-A/VEGFR2 signaling pathway.

Multiple members of the transforming growth factor beta (TGFβ) family of secreted factors play central inductive and patterning roles during embryogenesis. During gastrulation in vertebrates, the bone morphogenetic protein (BMP) sub-family is linked to formation of the embryonic organizer, Spemann's organizer in Xenopus, and dorsal-ventral mesoderm patterning. Our knowledge regarding the BMP receptors mediating this signaling is still very incomplete. The BMPR1A (ALK3) and BMPR1B (ALK6) receptors are known to mediate the BMP4 signal. These receptors belong to the ALK1 subfamily of type I receptors that also includes ACVR1 (ALK2), and ACVRL1 (ALK1). We studied by qPCR and in situ hybridization the spatio-temporal expression patterns of ALK2 and ALK1 and compared them to ALK3 and ALK6, and to the main BMPs expressed during gastrulation, i.e., BMP4, BMP7, BMP2, and ADMP, in an attempt to establish a link between ligands and receptors. There is extensive overlap between BMP4, and ALk3 and Alk6 expression, supporting their functional interaction. Robust Alk6 expression was observed from mid-gastrula. Animal region expression of both receptors shows co-expression with BMP4 and BMP7. Alk2 transcripts were detected within the organizer, overlapping with its proposed ligand, ADMP, suggesting a probable function within the organizer. Alk1 is very weakly expressed during gastrula, but its transcripts were localized to the lateral marginal zone flanking the organizer domain. No receptor closely matched the maternal BMP2 expression, although Alk2, Alk3, and Alk6, have transcripts of maternal origin. Our analysis shows that the BMP ligands and their receptors exhibit dynamic expression patterns during gastrula stages.

OBJECTIVE

To assess the expression of activin receptor-like kinase 1 (ALK1) and investigate its possible relationship with microvessel density (MVD) in different forms of oral lichen planus (OLP) compared to controls' biopsies.

METHODS

Biopsies from 20 reticular/papular OLP (R/PLP), 20 atrophic/erosive OLP (A/ELP) patients, and 20 healthy subjects were immunohistochemically analyzed and statistically compared and correlated for ALK1 expression and MVD as assessed by CD34 expression.

RESULTS

All OLP specimens revealed the presence of positive cytoplasmic CD34 immunostaining in endothelial cells, with statistically high significant MVD in each of R/PLP (Median; M = 4.40) and A/ELP (M = 7.69) compared to controls (M = 1.16) (P < 0.001). Statistically significant MVD was found in A/ELP compared to R/PLP (P < 0.001). All control specimens revealed negative ALK1 immunostaining of the few inflammatory cells found, while 85% of A/ELP cases and 70% of R/PLP cases showed positively immunostained sections for ALK-1, with statistically significant higher ALK1 expression In A/ELP (M = 1.95) compared to R/PLP (M = 0.86) (P = 0.005). No significant correlation between CD34 and ALK1 was detected in R/PLP (r = 0.081), while a barely moderate positive correlation was found in A/ELP (r = 0.396).

CONCLUSIONS

ALK1 expression and MVD are increased in OLP, particularly in A/ELP type.

Brain arteriovenous malformation (bAVM), characterized by tangled dysplastic vessels, is an important cause of intracranial hemorrhage in young adults, and its pathogenesis and progression are not fully understood. Patients with haploinsufficiency of transforming growth factor-β (TGF-β) receptors, activin receptor-like kinase 1 (ALK1) or endoglin (ENG) have a higher incidence of bAVM than the general population. However, bAVM does not develop effectively in mice with the same haploinsufficiency. The expression of integrin β8 subunit (ITGB8), another member in the TGF-β superfamily, is reduced in sporadic human bAVM. Brain angiogenic stimulation results at the capillary level of vascular malformation in adult Alk1 haploinsufficient (Alk1 +/- ) mice. We hypothesized that deletion of Itgb8 enhances bAVM development in adult Alk1 +/- mice. An adenoviral vector expressing Cre recombinase (Ad-Cre) was co-injected with an adeno-associated viral vector expressing vascular endothelial growth factor (AAV-VEGF) into the brain of Alk1 +/-;Itgb8-floxed mice to induce focal Itgb8 gene deletion and angiogenesis. We showed that compared with Alk +/- mice (4.75 ± 1.38/mm2), the Alk1 +/-;Itgb8-deficient mice had more dysplastic vessels in the angiogenic foci (7.14 ± 0.68/mm2, P = 0.003). More severe hemorrhage was associated with dysplastic vessels in the brain of Itgb8-deleted Alk1 +/- , as evidenced by larger Prussian blue-positive areas (1278 ± 373 pixels/mm2 vs. Alk1 +/- : 320 ± 104 pixels/mm2; P = 0.028). These data indicate that both Itgb8 and Alk1 are important in maintaining normal cerebral angiogenesis in response to VEGF. Itgb8 deficiency enhances the formation of dysplastic vessels and hemorrhage in Alk1 +/- mice.

Symmetry breaking by cellular polarization is an exquisite requirement for the cell-cycle of Saccharomyces cerevisiae cells, as it allows bud emergence and growth. This process is based on the formation of polarity clusters at the incipient bud site, first, and the bud tip later in the cell-cycle, that overall promote bud emission and growth. Given the extreme relevance of this process, a surveillance mechanism, known as the morphogenesis checkpoint, has evolved to coordinate the formation of the bud and cell cycle progression, delaying mitosis in the presence of morphogenetic problems. The atypical protein kinase haspin is responsible for histone H3-T3 phosphorylation and, in yeast, for resolution of polarity clusters in mitosis. Here, we report a novel role for haspin in the regulation of the morphogenesis checkpoint in response to polarity insults. Particularly, we show that cells lacking the haspin ortholog Alk1 fail to achieve sustained checkpoint activation and enter mitosis even in the absence of a bud. In alk1Δ cells, we report a reduced phosphorylation of Cdc28-Y19, which stems from a premature activation of the Mih1 phosphatase. Overall, the data presented in this work define yeast haspin as a novel regulator of the morphogenesis checkpoint in Saccharomyces cerevisiae, where it monitors polarity establishment and it couples bud emergence to the G2/M cell cycle transition.

Keywords: Saccharomyces cerevisiae; actin cytoskeleton; cell cycle; mitosis; morphogenesis checkpoint; polarization.

Hereditary Hemorrhagic Telangiectasia (HHT) is a rare disease, with an autosomal dominant inheritance and a worldwide incidence of about 1: 5000 individuals. In >80% of patients, HHT is caused by mutations in either ENG or ACVRL1, which code for ENDOGLIN and Activin A Receptor Type II-Like Kinase 1 (ALK1), belonging to the TGF-β/BMP signalling pathway. Typical HHT clinical features are mucocutaneous telangiectases, arteriovenous malformations, spontaneous and recurrent epistaxis, as well as gastrointestinal bleedings. An additional, but less frequent, clinical manifestation in some HHT patients is the presence of Pulmonary Arterial Hypertension (PAH). The aim of this work is to describe the functional role of a novel ENG intronic variant found in a patient affected by both HHT and PAH, in order to assess whether it has a pathogenic role. We proved that the variant lies in a novel binding-site for the transcription factor Sp1, known to be involved in the regulation of ENG and ACVRL1 transcription. We confirmed a pathogenic role for this intronic variant, as it significantly reduces ENG transcription by affecting this novel Sp1 binding-site.