Our investigation defines an overarching sequence of events that begins initially with a loss of endothelial cell polarity as a result of β1 integrin ablation. Once cell polarity is lost, the mechanisms that maintain a single endothelial layer, as well as the formation of a patent lumen, are also lost. Arrest of lumen formation can also be seen with β1 integrin pharmacological blockade, both in the avian embryo (Drake et al., 1992
) and the postnatal retina (). Endothelial deletion of β1 integrin results in substantial increases and ubiquitous localization of cell-cell adhesion proteins: Claudin-5, PECAM-1, VE-cadherin and CD99 (). This resembles patterns of abnormal lumen formation in Drosophila heart tubes, where gain of function E-cadherin mutants display increased cell adhesion and subsequent loss of luminization (Santiago-Martinez et al., 2008
). The data presented here suggests that at least within the endothelium, VE-cadherin, and other cell-cell adhesion molecules operate downstream of cell-matrix cues and internal cell polarity cues (Par3) to set up ordered polarized cell-cell contacts.
In models of in vitro endothelial lumen formation, events are initiated downstream of integrin-extracellular matrix signaling, but also require the activation of Cdc42, Rac1, Pak2/4 and the Par3/6/aPKC complex (Koh et al., 2008
). While we did not examine Cdc42 or other Rho family GTPases, the genetic deletion of β1 integrin may affect its ability to activate Cdc42. Yet, our data also suggests a genetic link between β1 integrin and Par3, as ablation of β1 integrin results in loss of Par3. Another contrast to the in vitro data is that while lumen formation of mid-sized arteries was blocked upon β1 integrin endothelial ablation, lumens of single cell capillaries were unaffected. An initial conclusion is that much like epithelial cells (Lubarsky and Krasnow, 2003
), lumen formation in vessels of distinct type and caliber is likely to employ alternative processes/molecules.
Recent data in epithelial tube formation suggests that there are Cdc42 independent mechanisms in forming a patent lumen, and that separate and distinct polarity complexes can function as polarity regulators: the association of Cdc42 with polarity complex members Par6 and aPKC being one complex and separately Par3 localized to tight junctions being another (Jaffe et al., 2008
; Martin-Belmonte et al., 2007
). Par6 and aPKC have been shown to interact independently of Par3 (Yamanaka et al., 2003
). This divergent set of events fits nicely with our data, as we did not observe any transcriptional changes in other polarity complex members, Par6 and aPKC included (Figure S3D
and data not shown). Specific to endothelium, Par3 can associate with VE-cadherin in absence of aPKC, and prior to the association of Par6 (Iden et al., 2006
). Thus, it may be that cell-cell and cell-matrix contacts operate separately in maintaining cell polarity through convergent pathways, where both cadherins and integrins are capable of activating Cdc42 pathways (Desai et al., 2009
; Koh et al., 2008
) or alternatively regulating polarity proteins. In our study, cell-matrix cues (via β1 integrin) appear to exert initial and critical control of cell-cell adhesion protein distribution and the polarity protein Par3.
In epithelial cells, successful lumen formation requires directed centripetal vacuole movement towards the future lumen, and eventual coalescence with the plasma membrane. When β1 integrin is deleted, endothelial cells are no longer polarized, and thus may become incapable of directed vacuole movement and fusion leading to the retention of vacuoles. This is a likely cause or consequence of the increase in Rab7, a Rab GTPase that may prove critical to endothelial vacuole trafficking; analagous to other Rabs during epithelial lumen formation (Desclozeaux et al., 2008
). While the downstream mechanisms of lumen formation (vacuole formation and fusion) are employed in β1 integrin ablated vessels, the phenotype is restricted to arterial endothelial cells. It is well known that sub-specification within endothelial lineages include identity markers for each endothelial subtype. Our study demonstrates that in addition to canonical arterial markers such as Dll4, Par3 (especially isoform 150kD) is preferentially expressed in nascent arterial vessels. However, the Par3 protein increase and isoform pattern in arteries is fleeting, and eventually becomes non-existent in the adult. The significance of specific Par3 isoform expression is unclear, but differences in isoform binding abilities have been previously shown (Sfakianos et al., 2007
). It is likely that arteries activate a specific and exclusive set of events for lumen formation that are then silenced after successful patency.
The downstream consequence of vascular β1 integrin ablation, as dissected in this study, lends mechanistic insight into the process of polarity maintenance and lumen formation in arterial endothelium. The first and main cause of these subsequent events appears to be loss of Par3 and thus cell polarity, which includes abnormal distribution of cell-cell adhesion molecules. As a result, the endothelial cells adopt atypical cell shapes and increase their adhesive properties. While intracellular vacuoles appear capable of forming in lieu of β1 integrin, they are no longer capable of directed migration and coalescence to form a lumen. However, lumen formation is restored with the addition of Par3 protein, suggesting that the adhesion changes and lumen defects are downstream of polarity cues, which are regulated by cell-matrix interactions via β1 integrin ().