When vessel constriction fails: Cellular mechanics linking developmental remodeling to vascular malformations

Blood vessels are important for supplying blood to tissues within the body with oxygen and nutrients as well as carrying away metabolic waste. To ensure efficient blood flow and tissue perfusion, tight regulation of blood vessel diameter is paramount. During development, blood vessels undergo extensive remodeling to acquire appropriate diameters and misregulation of this process can lead to vascular malformations seen in human pathologies, such as Cerebral Cavernous Malformations (CCM), a genetic disease characterized by clusters of abnormally dilated blood vessels. While changes in the number and shape of endothelial cells (ECs)—the building blocks of blood vessels—are recognized to be critical factors determining vessel diameter, the underlying mechanisms that coordinate these changes remain poorly understood.

Now a new multiscale study led by Research Scientist Yan Chen and Team Director Li-Kun Phng of the Laboratory for Vascular Morphogenesis at RIKEN BDR, in collaboration with the Laboratory for Physical Biology (Tatsuo Shibata, Team Director) and other colleagues, investigated blood vessel remodeling across subcellular, cellular and tissue scales using the arterial and venous intersegmental vessels (aISVs and vISVs) in zebrafish as a model. Using approaches such as high-resolution imaging, manipulation of actin cytoskeleton, in vivo laser ablation techniques and mathematical modeling, the team unveiled cellular and molecular-level mechanical mechanisms involved in regulating blood vessel diameter and demonstrated how their misregulation can lead to pathological vascular malformations seen in vascular diseases affecting blood flow. Their study was published in the online journal, Nature Communications.

Blood vessel remodeling takes place following vessel perfusion and can be observed after 2 days post-fertilization (dpf) in zebrafish embryos. The team first examined the temporal dynamics of ECs, particularly cell numbers and shape, in the ISVs during vessel remodeling using high-resolution time-lapse imaging. They discovered that contrary to reports by other groups of a positive correlation between increases in EC number and EC diameter during vessel remodeling, there was in fact an inverse relationship with EC numbers showing a significant increase while EC and vessel diameters decreased.

The team then investigated the potential role of cell shape in vessel remodeling by quantifying the two-dimensional temporal shape changes such as the area and aspect ratio of ECs making up the vessel. They found that there was a significant decrease in EC area between 2 to 4 dpf, with no changes to cell aspect ratio, indicating that cells reduced (contracted) in size in both circumferential (radial) and longitudinal (axial) axes. Quantitative strain analyses revealed that increase in EC numbers contributes to vessel elongation while EC contraction and rearrangement contributed to vessel diameter constriction.

The group next asked whether failure of ECs to undergo cell contractions can lead to vascular malformations seen in human pathologies such as CCM, a disease characterized by enlarged blood vessels in the brain that can lead to strokes. CCM studies using different model systems, including zebrafish, have revealed that mutations in CCM1/KRIT1 lead to enlarged ECs and vessels, but the underlying mechanisms have not been clarified. They confirmed that Krit1 was enriched at junctions and colocalized with circumferential actin bundles in ECs, suggesting that Krit1 anchors circumferential actin at cell junctions. Overexpression of Krit1 showed increased organization of circumferential actin along ISVs as well as enhanced vessel constriction. Contrastingly, in the absence of Krit1, actin was predominantly organized as mesh or longitudinal bundles, resulting in enlarged ECs and dilated vessels. These studies demonstrate that Krit1 regulates EC contraction and vessel size by ensuring the formation of circumferential actin in the cell cortex.

“Our multiscale study demonstrates how circumferential actomyosin drives contraction of ECs, which in turn regulate blood vessel diameter during development,” explained Chen. “We also show that disruption of the EC mechanics we uncovered can lead to phenotypes of blood vessel dilation seen in some human vascular diseases such as CCM.” 

Phng added, “Many people have identified the genetic mutations or signaling pathways affected in models of vascular malformations, but the cellular mechanisms had not clearly been shown. Our zebrafish ISV model of vessel remodeling has allowed us to perform high-resolution imaging in vivo, which has been difficult to carry out in other animal models, providing a host of new mechanistic insights into how blood vessel diameter is controlled in development and disease pathogenesis.”