Zhengda Sun

De Novo Cerebrovascular Malformation in the Adult Mouse After Endothelial Alk1 Deletion and Angiogenic Stimulation

Background and Purpose— In humans, activin receptor-like kinase 1 (Alk1) deficiency causes arteriovenous malformations (AVMs) in multiple organs, including the brain. Focal Alk1 pan-cellular deletion plus vascular endothelial growth factor stimulation induces brain AVMs in the adult mouse. We hypothesized that deletion of Alk1 in endothelial cell (EC) alone plus focal vascular endothelial growth factor stimulation is sufficient to induce brain AVM in the adult mouse. Methods— Focal angiogenesis was induced in the brain of 8-week-old Pdgfb-iCreER;Alk12f/2f mice by injection of adeno-associated viral vectors expressing vascular endothelial growth factor. Two weeks later, EC-Alk1 deletion was induced by tamoxifen treatment. Vascular morphology was analyzed, and EC proliferation and dysplasia index (number of vessels with diameter >15 &mgr;m per 200 vessels) were quantified 10 days after tamoxifen administration. Results— Tangles of enlarged vessels resembling AVMs were present in the brain angiogenic region of tamoxifen-treated Pdgfb-iCreER;Alk12f/2f mice. Induced brain AVMs were marked by increased dysplasia index (P<0.001) and EC proliferation clustered within the dysplastic vessels. AVMs were also detected around the ear tag-wound and in other organs. Conclusions— Deletion of Alk1 in EC in adult mice leads to an increased local EC proliferation during brain angiogenesis and de novo brain AVM.

Meta-Analysis of Public Microarray Datasets Reveals Voltage-Gated Calcium Gene Signatures in Clinical Cancer Patients

Voltage-gated calcium channels (VGCCs) are well documented to play roles in cell proliferation, migration, and apoptosis; however, whether VGCCs regulate the onset and progression of cancer is still under investigation. The VGCC family consists of five members, which are L-type, N-type, T-type, R-type and P/Q type. To date, no holistic approach has been used to screen VGCC family genes in different types of cancer. We analyzed the transcript expression of VGCCs in clinical cancer tissue samples by accessing ONCOMINE (www.oncomine.org), a web-based microarray database, to perform a systematic analysis. Every member of the VGCCs was examined across 21 different types of cancer by comparing mRNA expression in cancer to that in normal tissue. A previous study showed that altered expression of mRNA in cancer tissue may play an oncogenic role and promote tumor development; therefore, in the present findings, we focus only on the overexpression of VGCCs in different types of cancer. This bioinformatics analysis revealed that different subtypes of VGCCs (CACNA1C, CACNA1D, CACNA1B, CACNA1G, and CACNA1I) are implicated in the development and progression of diverse types of cancer and show dramatic up-regulation in breast cancer. CACNA1F only showed high expression in testis cancer, whereas CACNA1A, CACNA1C, and CACNA1D were highly expressed in most types of cancer. The current analysis revealed that specific VGCCs likely play essential roles in specific types of cancer. Collectively, we identified several VGCC targets and classified them according to different cancer subtypes for prospective studies on the underlying carcinogenic mechanisms. The present findings suggest that VGCCs are possible targets for prospective investigation in cancer treatment.

Persistent infiltration and pro-inflammatory differentiation of monocytes cause unresolved inflammation in brain arteriovenous malformation

An abnormally high number of macrophages are present in human brain arteriovenous malformations (bAVM) with or without evidence of prior hemorrhage, causing unresolved inflammation that may enhance abnormal vascular remodeling and exacerbate the bAVM phenotype. The reasons for macrophage accumulation at the bAVM sites are not known. We tested the hypothesis that persistent infiltration and pro-inflammatory differentiation of monocytes in angiogenic tissues increase the macrophage burden in bAVM using two mouse models and human monocytes. Mouse bAVM was induced through deletion of AVM causative genes, Endoglin (Eng) globally or Alk1 focally, plus brain focal angiogenic stimulation. An endothelial cell and vascular smooth muscle cell co-culture system was used to analyze monocyte differentiation in the angiogenic niche. After angiogenic stimulation, the Eng-deleted mice had fewer CD68+ cells at 2xa0weeks (Pxa0=xa00.02), similar numbers at 4xa0weeks (Pxa0=xa00.97), and more at 8xa0weeks (Pxa0=xa00.01) in the brain angiogenic region compared with wild-type (WT) mice. Alk1-deficient mice also had a trend toward more macrophages/microglia 8xa0weeks (Pxa0=xa00.064) after angiogenic stimulation and more RFP+ bone marrow-derived macrophages than WT mice (Pxa0=xa00.01). More CD34+ cells isolated from peripheral blood of patients with ENG or ALK1 gene mutation differentiated into macrophages than those from healthy controls (Pxa0<xa00.001). These data indicate that persistent infiltration and pro-inflammatory differentiation of monocytes might contribute to macrophage accumulation in bAVM. Blocking macrophage homing to bAVM lesions should be tested as a strategy to reduce the severity of bAVM.

Voltage-gated calcium channels: Novel targets for cancer therapy

Voltage-gated calcium channels (VGCCs) comprise five subtypes: The L-type; R-type; N-type; P/Q-type; and T-type, which are encoded by α1 subunit genes. Calcium ion channels also have confirmed roles in cellular functions, including mitogenesis, proliferation, differentiation, apoptosis and metastasis. An association between VGCCs, a reduction in proliferation and an increase in apoptosis in prostate cancer cells has also been reported. Therefore, in the present study, the online clinical database Oncomine was used to identify the alterations in the mRNA expression level of VGCCs in 19 cancer subtypes. Overall, VGCC family genes exhibited under-expression in numerous types of cancer, including brain, breast, kidney and lung cancers. Notably, the majority of VGCC family members (CACNA1C, CACNA1D, CACNA1A, CACNA1B, CACNA1E, CACNA1H and CACNA1I) exhibited low expression in brain tumors, with mRNA expression levels in the top 1–9% of downregulated gene rankings. A total of 5 VGCC family members (CACNA1A, CACNA1B, CACNA1E, CACNA1G and CACNA1I) were under-expressed in breast cancer, with a gene ranking in the top 1–10% of the low-expressed genes compared with normal tissue. In kidney and lung cancers, CACNA1S, CACNA1C, CACNA1D, CACNA1A and CACNA1H exhibited low expression, with gene rankings in the top 1–8% of downregulated genes. In conclusion, the present findings may contribute to the development of new cancer treatment approaches by identifying target genes involved in specific types of cancer.

High Throughput Identification of Monoclonal Antibodies to Membrane Bound and Secreted Proteins Using Yeast and Phage Display

Antibodies are ubiquitous and essential reagents for biomedical research. Uses of antibodies include quantifying proteins, identifying the temporal and spatial pattern of expression in cells and tissue, and determining how proteins function under normal or pathological conditions. Specific antibodies are only available for a small portion of the proteome, limiting study of those proteins for which antibodies do not exist. The technologies to generate target-specific antibodies need to be improved to obtain high quality antibodies to the proteome at reasonable cost. Here we show that renewable, validated, and standardized monoclonal antibodies can be generated at high throughput, without the need for antigen production or animal immunizations. In this study, 60 protein domains from 24 selected secreted proteins were expressed on the surface of yeast and used for selection of phage antibodies, over 400 monoclonal antibodies were identified within 3 weeks. A subset of these antibodies was validated for binding to cancer cells that overexpress the target protein by flow cytometry or immunohistochemistry. This approach will be applicable to many of the membrane-bound and the secreted proteins, 20–40% of the proteome, accelerating the timeline for Ab generation while reducing the cost.

Soluble FLT1 Gene Therapy Alleviates Brain Arteriovenous Malformation Severity

Background and Purpose— Brain arteriovenous malformation (bAVM) is an important risk factor for intracranial hemorrhage. Current therapies are associated with high morbidities. Excessive vascular endothelial growth factor has been implicated in bAVM pathophysiology. Because soluble FLT1 binds to vascular endothelial growth factor with high affinity, we tested intravenous delivery of an adeno-associated viral vector serotype-9 expressing soluble FLT1 (AAV9-sFLT1) to alleviate the bAVM phenotype. Methods— Two mouse models were used. In model 1, bAVM was induced in R26CreER;Eng2f/2f mice through global Eng gene deletion and brain focal angiogenic stimulation; AAV2-sFLT02 (an AAV expressing a shorter form of sFLT1) was injected into the brain at the time of model induction, and AAV9-sFLT1, intravenously injected 8 weeks after. In model 2, SM22&agr;Cre;Eng2f/2f mice had a 90% occurrence of spontaneous bAVM at 5 weeks of age and 50% mortality at 6 weeks; AAV9-sFLT1 was intravenously delivered into 4- to 5-week-old mice. Tissue samples were collected 4 weeks after AAV9-sFLT1 delivery. Results— AAV2-sFLT02 inhibited bAVM formation, and AAV9-sFLT1 reduced abnormal vessels in model 1 (GFP versus sFLT1: 3.66±1.58/200 vessels versus 1.98±1.29, P<0.05). AAV9-sFLT1 reduced the occurrence of bAVM (GFP versus sFLT1: 100% versus 36%) and mortality (GFP versus sFLT1: 57% [12/22 mice] versus 24% [4/19 mice], P<0.05) in model 2. Kidney and liver function did not change significantly. Minor liver inflammation was found in 56% of AAV9-sFLT1–treated model 1 mice. Conclusions— By applying a regulated mechanism to restrict sFLT1 expression to bAVM, AAV9-sFLT1 can potentially be developed into a safer therapy to reduce the bAVM severity.

Systematic analysis of the achaete-scute complex-like gene signature in clinical cancer patients

The achaete-scute complex-like (ASCL) family, also referred to as ‘achaete-scute complex homolog’ or ‘achaete-scute family basic helix-loop-helix transcription factor’, is critical for proper development of the nervous system and deregulation of ASCL plays a key role in psychiatric and neurological disorders. The ASCL family consists of five members, namely ASCL1, ASCL2, ASCL3, ASCL4 and ASCL5. The ASCL1 gene serves as a potential oncogene during lung cancer development. There is a correlation between increased ASCL2 expression and colon cancer development. Inhibition of ASCL2 reduced cellular proliferation and tumor growth in xenograft tumor experiments. Although previous studies demonstrated involvement of ASCL1 and ASCL2 in tumor development, little is known on the remaining ASCL family members and their potential effect on tumorigenesis. Therefore, a holistic approach to investigating the expression of ASCL family genes in diverse types of cancer may provide new insights in cancer research. In this study, we utilized a web-based microarray database (Oncomine; www.oncomine.org) to analyze the transcriptional expression of the ASCL family in clinical cancer and normal tissues. Our bioinformatics analysis revealed the potential involvement of multiple ASCL family members during tumor onset and progression in multiple types of cancer. Compared to normal tissue, ASCL1 exhibited a higher expression in cancers of the lung, pancreas, kidney, esophagus and head and neck, whereas ASCL2 exhibited a high expression in cancers of the breast, colon, stomach, lung, head and neck, ovary and testis. ASCL3, however, exhibited a high expression only in breast cancer. Interestingly, ASCL1 expression was downregulated in melanoma and in cancers of the bladder, breast, stomach and colon. ASCL2 exhibited low expression levels in sarcoma, melanoma, brain and prostate cancers. Reduction in the expression of ASCL3 was detected in lymphoma, bladder, cervical, kidney and epithelial cancers. Similarly, ASCL5 exhibited low expression in the majority of brain cancer subtypes, such as glioblastoma and oligodendroglioma. This analysis supports the hypothesis that specific ASCL members may play an important role in cancer development. Collectively, our data suggest that alterations in the expression of ASCL gene family members are correlated with cancer development. Furthermore, ASCL family members were categorized according to cancer subtype. The aim of this report was to provide novel insights to the significance of the ASCL family in various cancers and our findings suggested that the ASCL gene family may be an ideal target for future cancer studies.

Endovascular biopsy: Technical feasibility of novel endothelial cell harvesting devices assessed in a rabbit aneurysm model

The lack of safe and reliable methods to sample vascular tissue in situ limits discovery of the underlying genetic and pathophysiological mechanisms of many vascular disorders, including aneurysms. We investigated the feasibility and comparable efficacy of in vivo vascular endothelial cell sampling using a spectrum of endovascular devices. Using the rabbit elastase carotid aneurysm model we evaluated the performance of existing aneurysmal coils, intracranial stents, and stent-like devices to collect vascular endothelial cells. Additionally, we modified a subset of devices to assess the effects of alterations to coil pitch, coil wire contour, and stent surface finishing. Device performance was evaluated by (1) the number of viable endothelial cells harvested, (2) the degree of vascular wall damage analyzed using digital subtraction angiography and histopathological analysis, and (3) the ease of device navigability and retrieval. Isolated cells underwent immunohistochemical analysis to confirm cell type and viability. Coil and stent specifications, technique, and endothelial cell counts were tabulated and statistical analysis performed. Using conventional detachable-type and modified aneurysm coils 11 of 14 (78.6%) harvested endothelial cells with a mean of 7.93 (±8.33) cells/coil, while 15 of 15 (100%) conventional stents, stent-like devices and modified stents harvested endothelial cells with a mean of 831.33 (±887.73) cells/device. Coil stiffness was significantly associated with endothelial cell count in univariate analysis (pu2009=u20090.044). For stents and stent-like devices univariate analysis demonstrated stent-to-aorta diameter ratios (pu2009=u20090.001), stent length (pu2009=u20090.049), and the use of a pulling retrieval technique (pu2009=u20090.019) significantly predictive of endothelial cell counts, though a multivariate model using these variables demonstrated only the stent-to-aorta diameter ratio (pu2009=u20090.029) predictive of endothelial cell counts. Modified devices did not significantly impact harvesting. The efficacy and safety of existing aneurysm coils, intracranial stents and stent-like devices in collecting viable endothelial cells was confirmed. The technique is reproducible and the quantity and quality of collected endothelial cells is adequate for targeted genetic analysis.

Endovascular biopsy: Strategy for analyzing gene expression profiles of individual endothelial cells obtained from human vessels(✩).

Graphical abstract

Endovascular Biopsy: Evaluating the Feasibility of Harvesting Endothelial Cells Using Detachable Coils

The absence of safe and reliable methods to harvest vascular tissue in situ limits the discovery of the underlying genetic and pathophysiological mechanisms of many vascular disorders such as aneurysms. We investigated the feasibility and comparable efficacy of endothelial cell collection using a spectrum of endovascular coils. Nine detachable coils ranging in k coefficient (0.15–0.24), diameter (4.0 mm–16.0 mm), and length (8.0 cm–47.0 cm) were tested in pigs. All coils were deployed and retrieved within the iliac artery of pigs (three coils/pig). Collected coils were evaluated under light microscopy. The total and endothelial cells collected by each coil were quantified. The nucleated cells were identified by Wright-Giemsa and DAPI stains. Endothelial and smooth muscle cells were identified by CD31 and α-smooth muscle actin antibody staining. Coils were deployed and retrieved without technical difficulty. Light microscopy demonstrated sheets of cellular material concentrated within the coil winds. All coils collected cellular material while five of nine (55.6%) coils retrieved endothelial cells. Coils collected mean endothelial cell counts of 89.0±101.6. Regression analysis demonstrated a positive correlation between increasing coil diameter and endothelial cell counts (R2 = 0.52, p = 0.029). Conventional detachable coils can be used to harvest endothelial cells. The number of endothelial cells collected by a coil positively correlated with its diameter. Given the widespread use of coils and their well-described safety profile their potential as an endovascular biopsy device would expand the availability of tissue for cellular and molecular analysis.