Role of the cytoskeleton in formation and maintenance of angiogenic sprouts

Abstract

Angiogenesis is the formation of new blood vessels from pre-existing structures, and is a key step in tissue and organ development, wound healing and pathological events. Changes in cell shape orchestrated by the cytoskeleton are integral to accomplishing the various steps of angiogenesis, and an intact cytoskeleton is also critical for maintaining newly formed structures. This review focuses on how the 3 main cytoskeletal elements--microfilaments, microtubules, and intermediate filaments--regulate the formation and maintenance of angiogenic sprouts. Multiple classes of compounds target microtubules and microfilaments, revealing much about the role of actin and tubulin and their associated molecules in angiogenic sprout formation and maintenance. In contrast, intermediate filaments are much less studied, yet intriguing evidence suggests a vital, but unresolved, role in angiogenic sprouting. This review discusses evidence for regulatory molecules and pharmacological compounds that affect actin, microtubule and intermediate filament dynamics to alter various steps of angiogenesis, including endothelial sprout formation and maintenance.

Fig. 1

Immunofluorescence analysis of tubulin, vimentin and actin arrangement in 2-D versus 3-D cultures. Top row: Human umbilical vein endothelial cells (passage 5) were seeded on 18-mm circular coverslips coated with collagen type I (20 μg/ml) and allowed to attach overnight. Cells were treated with 1 μM S1P for 1 h before being fixed and processed for immunofluorescence as described in the ‘Appendix’. Primary antibodies utilized were anti-α-tubulin (clone DMA1; Sigma-Aldrich; 1:100), anti-vimentin [V9 FL (p) epitope; Santa Cruz; 1:100] and anti-actin (AB-1 clone JLA20; EMD Biosciences; 1:25). Images were collected with a Nikon Eclipse TE2000-U microscope. ×60. Bottom row: Cells were seeded on 3-D collagen type I matrices (2.5 mg/ml) with growth factors and 1 μM S1P for 24 h, fixed in 4% paraformaldehyde in PBS for 30 min and processed for immunofluorescence. Images collected with a Zeiss Imager. A1m confocal microscope (0.5- to 1.5-μm stacks that were layered into 1 image). ×40. Arrowheads indicate extended peripheral processes.

Fig. 2

Schematic depicting microtubule, vimentin and actin localization in quiescent versus activated endothelial cells: quiescent endothelial cells (a) and sprouting endothelial cells (b). Placement is based on the data shown in figure 1.

Fig. 3

Schematic illustration illustrating key steps in angiogenesis, along with molecules that transduce signals to the cytoskeleton to induce sprouting angiogenesis. a Quiescent endothelium exhibiting intact basement membrane (red), mural cell (yellow) and intact junctions. b Sprout initiation is stimulated by local production of angiogenic factors, which disrupt junctions and basement membrane integrity to initiate sprouting responses. c Sprout extension and new vessel growth. Key molecules that control lumen formation, junctional signaling and fine protrusion formation are indicated.