Se-woon Choe

Real-time imaging of de novo arteriovenous malformation in a mouse model of hereditary hemorrhagic telangiectasia

Arteriovenous malformations (AVMs) are vascular anomalies where arteries and veins are directly connected through a complex, tangled web of abnormal arteries and veins instead of a normal capillary network. AVMs in the brain, lung, and visceral organs, including the liver and gastrointestinal tract, result in considerable morbidity and mortality. AVMs are the underlying cause of three major clinical symptoms of a genetic vascular dysplasia termed hereditary hemorrhagic telangiectasia (HHT), which is characterized by recurrent nosebleeds, mucocutaneous telangiectases, and visceral AVMs and caused by mutations in one of several genes, including activin receptor-like kinase 1 (ALK1). It remains unknown why and how selective blood vessels form AVMs, and there have been technical limitations to observing the initial stages of AVM formation. Here we present in vivo evidence that physiological or environmental factors such as wounds in addition to the genetic ablation are required for Alk1-deficient vessels to develop to AVMs in adult mice. Using the dorsal skinfold window chamber system, we have demonstrated for what we believe to be the first time the entire course of AVM formation in subdermal blood vessels by using intravital bright-field images, hyperspectral imaging, fluorescence recordings of direct arterial flow through the AV shunts, and vascular casting techniques. We believe our data provide novel insights into the pathogenetic mechanisms of HHT and potential therapeutic approaches.

3D In Vivo optical coherence tomography based on a low-voltage, large-scan-range 2D MEMS mirror

3D in vivo optical imaging on a mouse has been obtained using a 2D MEMS mirror for lateral scanning in a time-domain optical coherence tomography (OCT) system. The MEMS mirror aperture size is 1 x 1 mm(2), and the device footprint is 2 x 2 mm(2). The MEMS mirror scans +/- 30 degrees optical angles about both x and y-axis at only 5.5V DC voltage. An endoscopic probe with an outer diameter of 5.8 mm has been designed, manufactured and packaged. The probe scans an average transverse area of 2 mm x 2 mm. The imaging speed of the probe is about 2.5 frames per second, limited by the speed of the employed optical delay line.

VEGF neutralization can prevent and normalize arteriovenous malformations in an animal model for hereditary hemorrhagic telangiectasia 2

Arteriovenous malformation (AVM) refers to a vascular anomaly where arteries and veins are directly connected through a complex, tangled web of abnormal AV fistulae without a normal capillary network. Hereditary hemorrhagic telangiectasia (HHT) types 1 and 2 arise from heterozygous mutations in endoglin (ENG) and activin receptor-like kinase 1 (ALK1), respectively. HHT patients possess AVMs in various organs, and telangiectases (small AVMs) along the mucocutaneous surface. Understanding why and how AVMs develop is crucial for developing therapies to inhibit the formation, growth, or maintenance of AVMs in HHT patients. Previously, we have shown that secondary factors such as wounding are required for Alk1-deficient vessels to develop skin AVMs. Here, we present evidences that AVMs establish from nascent arteries and veins rather than from remodeling of a preexistent capillary network in the wound-induced skin AVM model. We also show that VEGF can mimic the wound effect on skin AVM formation, and VEGF-neutralizing antibody can prevent skin AVM formation and ameliorate internal bleeding in Alk1-deficient adult mice. With topical applications at different stages of AVM development, we demonstrate that the VEGF blockade can prevent the formation of AVM and cease the progression of AVM development. Taken together, the presented experimental model is an invaluable system for precise molecular mechanism of action of VEGF blockades as well as for preclinical screening of drug candidates for epistaxis and gastrointestinal bleedings.

Common and Distinctive Pathogenetic Features of Arteriovenous Malformations in Hereditary Hemorrhagic Telangiectasia 1 and Hereditary Hemorrhagic Telangiectasia 2 Animal Models—Brief Report

Objective— Hereditary hemorrhagic telangiectasia is a genetic disorder characterized by visceral and mucocutaneous arteriovenous malformations (AVMs). Clinically indistinguishable hereditary hemorrhagic telangiectasia 1 and hereditary hemorrhagic telangiectasia 2 are caused by mutations in ENG and ALK1, respectively. In this study, we have compared the development of visceral and mucocutaneous AVMs in adult stages between Eng- and Alk1-inducible knockout (iKO) models. Approach and Results— Eng or Alk1 were deleted from either vascular endothelial cells (ECs) or smooth muscle cells in adult stages using Scl-CreER and Myh11-CreER lines, respectively. Latex perfusion and intravital spectral imaging in a dorsal skinfold window chamber system were used to visualize remodeling vasculature during AVM formation. Global Eng deletion resulted in lethality with visceral AVMs and wound-induced skin AVMs. Deletion of Alk1 or Eng in ECs, but not in smooth muscle cells, resulted in wound-induced skin AVMs. Visceral AVMs were observed in EC-specific Alk1-iKO but not in Eng-iKO. Intravital spectral imaging revealed that Eng-iKO model exhibited more dynamic processes for AVM development when compared with Alk1-iKO model. Conclusions— Both Alk1- and Eng-deficient models require a secondary insult, such as wounding, and ECs are the primary cell type responsible for the pathogenesis. However, Alk1 but not Eng deletion in ECs results in visceral AVMs.

A 2.8-mm Imaging Probe Based On a High-Fill-Factor MEMS Mirror and Wire-Bonding-Free Packaging for Endoscopic Optical Coherence Tomography

This paper reports a miniature optical coherence tomography (OCT) probe and high-resolution 3D OCT imaging results obtained with this probe. The probe is only 2.8-mm in diameter, enabled by a unique high-fill-factor electrothermal MEMS mirror with hidden actuators and a novel wire-bonding-free (WBF) packaging technique. The MEMS mirror has a large mirror aperture of 1 mm with a chip size of only 1.55×1.7×0.5 mm3. The fabricated device achieves large 2-D scan optical angles up to 46° at only 4.8 V. High-resolution 3D OCT imaging results are also demonstrated using this assembled probe.

Enhanced responses to angiogenic cues underlie the pathogenesis of hereditary hemorrhagic telangiectasia 2.

Hereditary Hemorrhagic Telangiectasia (HHT) is a genetic vascular disease in which arteriovenous malformations (AVMs) manifest in skin and multiple visceral organs. HHT is caused by heterozygous mutations in endoglin (ENG), activin receptor-like kinase 1 (ALK1), or SMAD4. ALK1 regulates angiogenesis, but the precise function of ALK1 in endothelial cells (ECs) remains elusive. Since most blood vessels of HHT patients do not produce pathological vascular lesions, ALK1 heterozygous ECs may be normal unless additional genetic or environmental stresses are imposed. To investigate the cellular and biochemical phenotypes of Alk1-null versus Alk1-heterozygous ECs, we have generated pulmonary EC lines in which a genotype switch from the Alk1-conditional allele (Alk1 2f) to the Alk1-null allele (Alk1 1f) can be induced by tamoxifen treatment. Alk1-null (1 f/1 f) ECs displayed increased migratory properties in vitro in response to bFGF compared with Alk1-het (2 f/1 f) ECs. The 1 f/1 f-ECs formed a denser and more persistent tubular network as compared with their parental 2 f/1 f-ECs. Interestingly, the response to BMP-9 on SMAD1/5 phosphorylation was impaired in both 2 f/1 f- and 1 f/1 f-ECs at a comparable manner, suggesting that other factors in addition to SMADs may play a crucial role for enhanced angiogenic activity in 1 f/1 f-ECs. We also demonstrated in vivo that Alk1-deficient ECs exhibited high migratory and invasive properties. Taken together, these data suggest that enhanced responses to angiogenic cues in ALK1-deficient ECs underlie the pathogenesis of HHT2.

Intravital microscopy imaging of macrophage localization to immunogenic particles and co-localized tissue oxygen saturation.

Well-designed biomaterial polymer particle-based vaccines will optimally promote immune cell antigen-presenting behavior while minimizing adverse inflammatory responses to the particles and encapsulated drugs or adjuvants. It is important in the design of particle-based vaccines to consider possible harmful effects of immune response on tissue at the vaccination site. Intravital microscopy with rodent dorsal skin window chambers enables in vivo serial observations in the same animal, and such models which have been used to study angiogenesis and macrophage response to implanted biomaterials may also be useful for the development of particle-based vaccines. To our knowledge there have been no reports where intravital microscopy has documented real-time immune cell localization and potentially harmful co-localized tissue effects. In this proof-of-principle study we used fluorescence and spectral imaging intravital microscopy of mouse window chambers to measure macrophage localization and co-localized tissue microvessel hemoglobin saturation changes in response to an immunogenic stimulus from polymer particles loaded with lipopolysaccharide (LPS) serving as a model vaccine/adjuvant system. We observed greater and faster macrophage localization to stronger inflammatory stimuli from LPS-loaded particle doses, a trend of decreased microvessel oxygenation with increased macrophage accumulation and, in an extreme case, complete microvessel collapse accompanied by tissue necrosis. Our technique may be useful for optimizing design of particle-based vaccines and may give insight into the use of hemoglobin saturation as a biomarker of tissue inflammation for clinical investigations of particle-based vaccines.

Selective effects of oral antiangiogenic tyrosine kinase inhibitors on an animal model of hereditary hemorrhagic telangiectasia

Essentials Antiangiogenic drugs are indicated as therapies for hereditary hemorrhagic telangiectasia. We interrogated the response to four antiangiogenic drugs for anemia and intestinal bleeding. Sorafenib and a pazopanib analog significantly improved while erlotinib worsened anemia. Some oral antiangiogenic drugs were effective in reducing intestinal bleeding.

A 2.8-MM imaging probe based on a high-fill-factor MEMS mirror and wire-bonding-free packaging for endoscopic optical coherence tomography

This paper reports a miniature optical coherence tomography (OCT) probe and high-resolution 3-D OCT imaging results obtained with this side-view probe. The probe is only 2.8 mm in diameter, enabled by a unique high-fill-factor electrothermal MEMS mirror with hidden actuators and a novel wire-bonding-free packaging technique. The MEMS mirror has a large mirror aperture of 1 mm with a chip size of only 1.55 mm × 1.7 mm × 0.5 mm. The fabricated device achieves large 2-D scan optical angles up to 46° at only 4.8 V. The specific time-domain OCT system utilized is detailed, and the assembled side-view probe demonstrates multiple high-resolution 3-D OCT imaging results that demonstrate detailed images of a mouse ear and images detecting the presence of tumor cells, and the contrast with a normal tissue is qualitatively analyzed.

In Vivo 3D and Doppler OCT Imaging Using Electrothermal MEMS Scanning Mirrors

Most cancers occur inside human body, so endoscopic high-resolution imaging modalities are required for early cancer detection and surgical removal. This paper reports in vivo endoscopic 3D imaging based on optical coherence tomography (OCT). Endoscopic imaging is enabled by integrating rapid-scanning MEMS mirror into a miniature imaging probe. The MEMS mirror has an aperture size of 1 mm by 1 mm and a chip size of 2 mm by 2 mm. The optical scan angle exceeds ±25 V at 6 Vdc, and thus large, constant-velocity, linear scan can be realized. The outer diameter of the probe is only 5 mm. The axial resolution is about 10 μm and the imaging speed is 2.5 frames per second. Doppler OCT imaging has also been demonstrated.