Mahtab Bahramsoltani

Intussusceptive Angiogenesis: A Biologically Relevant Form of Angiogenesis

Angiogenesis, i.e. the development and growth of blood vessels, is a major topic of research as it plays an important role in normal development and in various pathologies. Recent evidence revealed the existence of different mechanisms of blood vessel growth, including sprouting and intussusceptive angiogenesis, vascular mimicry, and blood vessel cooption. The latter two have only been observed in tumor growth, but sprouting and intussusceptive angiogenesis also occur in healthy, physiologically growing tissues. Despite this variety of angiogenic mechanisms, most of the current research is focused on the mechanism of sprouting angiogenesis because this mechanism was first described and because most existing experimental models are related to sprouting angiogenesis. Consequently, the mechanism of intussusceptive angiogenesis is often overlooked in angiogenesis research. Here, the mechanism of intussusceptive angiogenesis is reviewed and the current techniques and models for investigating intussusceptive angiogenesis are summarized. In addition, other mechanisms of vascular growth are briefly reviewed.

Quantitation of angiogenesis in vitro induced by VEGF-A and FGF-2 in two different human endothelial cultures - an all-in-one assay.

Angiogenic therapy is considered to be a promising tool for treatment of ischemic diseases. Many in vivo and in vitro assays have been developed to identify potential proangiogenic drugs and to investigate their mode of action. However, until now no validated system exists that would allow quantitation of angiogenesis in vitro in only one assay. Here, a previously established all-in-one in vitro assay based on staging of the angiogenic cascade was validated by quantitation of the effects of the known proangiogenic factors VEGF-A and FGF-2. Both growth factors were applied separately or in combination to human endothelial cell cultures derived from the heart and the foreskin, and angiogenesis was quantitated over 30 days of culture. Additionally, gene expression of VEGFR-1, VEGFR-2 and FGFR-1 at 3, 10, 20 or 40 days of cultivation was quantitated by RT-qPCR. In both cultures, VEGF-A as well as FGF-2 induced a run through all defined stages of angiogenesis in vitro. Application of VEGF-A only led to formation of irregular globular endothelial structures, while FGF-2 resulted in development of regular capillary-like structures. Quantitation of the angiogenic effects of VEGF-A and transcripts of VEGFR-1 and VEGFR-2 showed that a high VEGFR-1/VEGFR-2 ratio evoked deceleration of angiogenesis.

Angiogenesis and Collagen Type IV Expression in Different Endothelial Cell Culture Systems

In vitro angiogenesis assays constitute an important tool for studying the mechanisms of angiogenesis and for identification of pro‐ and anti‐angiogenic substances. Therefore, endothelial cell and media systems used for in vitro angiogenesis assays are required to mimic the angiogenic process in vivo including endothelial capability to express collagen type IV as a component of the basement membrane. In this study, the expression of collagen type IV and its α chains (α1‐6) was investigated in different endothelial cell culture systems in vitro qualitatively and quantitatively. These systems included four different batches of microvascular endothelial cells derived from the human skin, heart and lung, from which only two batches were found to be angiogenic and two batches were classified as non‐angiogenic. Distribution of the transcripts of the α chains of collagen type IV was similar in all cell and media systems investigated. However, secretion and deposition of a stable extracellular network of collagen type IV could only be observed in the angiogenic cultures. In conclusion, the consecutive steps of the angiogenic cascade in vivo as well as in vitro depend on an increasing secretion and subsequent extracellular deposition of collagen type IV.

Angiopoietins differentially influence in vitro angiogenesis by endothelial cells of different origin

Angiopoietins are important growth factors for vascular development and quiescence. They are promising targets for pro- or anti-angiogenic therapies in diverse pathologies, but the mechanisms of the ANGPT/TIE2 system are complex and not well understood. In the present study, the separate and combined effects of angiopoietin 1 and angiopoietin 2 were studied, using a recently developed in vitro angiogenesis model that allows both a quantitative and qualitative evaluation of the angiogenic cascade. This cell culture model was performed with microvascular endothelial cells (ECs) originating from different vascular beds, i.e. dermal ECs and cardiac ECs. In addition, the expression of the angiopoietins and the receptors, TIE1 and TIE2 was analyzed with RT-qPCR. This study revealed that the angiopoietins provoked a differential response in the two endothelial cultures. Both angiopoietin 1 as well as angiopoietin 2 elicited an angiogenic cascade in the dermal ECs but not in the cardiac ECs. In addition, the RT-qPCR data revealed marked differences in the endogenous expression pattern of these factors, indicating that the origin of endothelial cells might have an important impact on their angiogenic potential.

In vitro angiogenic potency in human microvascular endothelial cells derived from myocardium, lung and skin

Human microvascular endothelial cells derived from myocardium (HCMEC), lung (HPMEC) and foreskin (HDMEC) showed different angiogenic potency when cultivated in their original growth media provided by the distributors. In order to standardize microenvironmental conditions in an all-in-one assay of angiogenesis the aim of this study was to find one optimal growth medium for the endothelial cells derived from the different organs. Therefore each endothelial cell type was cultivated under identical conditions in the different original growth media as well as in several media formulations of the original growth media. Results reveal that even if cultivated in the same growth medium under exactly the same cultivation conditions--over a prolonged time period of 60 days--the endothelial cells still showed different angiogenic potency. This is due to a combination of extrinsic factors, i.e. the isolation procedure and in particular the growth medium, as well as to intrinsic differences between cells of diverse origin.

Searching for markers to identify angiogenic endothelial cells: A proteomic approach

In the field of angiogenesis research considerable effort is put in the development of in vitro assays of angiogenesis to replace animal experiments. Unfortunately, reproducibility of these assays frequently fails depending on the particular batch of endothelial cells delivered by the distributor. This is due to the lack of reliable markers for the identification and isolation of angiogenic microvascular endothelial cells that have the capacity to perform all stages of the angiogenic cascade. This study was carried out to identify potential markers for angiogenic versus non-angiogenic endothelial cells. The protein expression profile of four capillary-derived human microvascular primary endothelial cell cultures of which only two batches could be stimulated to angiogenesis was investigated and compared by two-dimensional gel electrophoresis. Seven proteins were found to be expressed in the angiogenic batches only. One protein was detected exclusively in the non-angiogenic batches. These proteins might be verified as markers for angiogenic endothelial cells.

Parapagus Conjoined Twin Calf: A Case Study – Focused on CT and Cardiac Abnormalities

Congenital duplication anomalies occasionally occur in both humans and animals. Although various forms of classification of these conjoined twins exist, each case should be considered as an individual. In the case study presented a Holstein Frisian calf, born alive after 281 days of normal gravidity was investigated by computed tomography (CT) and subsequent dissection. The calf could be classified as a parapagus dicephalus tetrabrachius. It exhibited two heads each of them with a complete cervical spine leading to a complete thoracic and lumbar spine and separate tails. Looking at the point of fusion, the twin was conjoined in the ventrolateral thoracic part of the body. The calf had two thoraces with four forelimbs and two pelvic limbs on a single pelvis. As two heads and thoraces were present, beginning at the oral cavity, two intestinal systems were observable leading to a fusion point at the ascending part of the duodenum been continued caudally as a single system. Within each thorax, two lungs and a heart were present. However, only the heart in the left thorax was normal shape, exhibiting a strong myocardium and increased size. In contrast, the heart within the right thorax was considerably smaller, round shaped, and it appeared to be spongy and not fully developed during the foetal period. Commonly, classification of conjoined twins is only based on the appearance of the skeletal system. However, in the case presented, the point of fusion of the skeletal system did not allow conclusions regarding the intestinal or cardiovascular system.

Quantitation of Tumor Angiogenesis In Vitro: An All-In-One Angiogenesis Assay

In vitro angiogenesis systems enable the analysis of pro- or anti-angiogenic compounds. Most in vitro models do not reproduce the entire angiogenic cascade, from cell migration and proliferation to tube formation. Here, we describe an all-in-one angiogenesis assay that mimics the entire angiogenic cascade in vitro, rendering this model an ideal tool for the in vitro testing.