23
about a 50% decrease in pericyte coverage of vessels [16]. Of interest is that these
mice show primary defects in microvessel beds (where pericyte coverage is present), while larger vessels are much less affected. This microvascular deficiency
phenotype manifests particularly in the kidney and central nervous system which
strongly resembles that observed in diabetic microangiopathy [16]. Loss of pericytes is a major pathogenic cause of this type of microvascular disease [51]. It is
important to further understand the signaling mechanisms which underlie pericyte
recruitment to EC-lined tubes, to both identify other factors that regulate this
recruitment as well as understand how pericytes invade 3D matrices to recruit to
these tubes. This is currently a major research direction of our laboratory.
1.3.14 Pericyte Recruitment to EC-Lined Tubes Stimulates
ECM Remodeling Events and Vascular Basement
Membrane Matrix Assembly
Using our new model of EC-pericyte tube co-assembly, we sought to identify how
pericytes contribute to vascular tube maturation and stabilization events. At different time points of tube co-assembly, we performed transmission electron microscopy and immunofluorescence microscopy to examine if basement membrane
matrix assembly occurred [94]. In Fig. 1.5b, we show immunostaining for the EC
marker CD31, while the pericytes stably express green fluorescent protein (GFP).
This image shows EC tube networks that have associated pericytes at day 5 of culture. We also show a light microscopy image of the coculture system in Fig. 1.5a.
As shown in Fig. 1.5c, d, there is marked deposition of laminin and collagen type
IV, two critical basement membrane matrix components. In addition, we reported
that fibronectin, nidogen-1, nidogen-2, and perlecan were also deposited around
EC-lined tubes only when pericytes were cocultured with ECs [94] (Fig. 1.5e). We
demonstrated that basement membrane matrices were observed by transmission
electron microscopy, only when EC-pericytes were cocultured [94], and over many
years we have never observed basement membrane deposition in the absence of
pericytes in electron microscopic studies. Further, we confirmed our results in vivo
and demonstrated that pericyte recruitment to developing quail EC tubes directly
correlates with vascular basement membrane assembly, at day 7 of embryonic
development [94]. Prior to pericyte recruitment, no vascular basement membranes
around EC tubes were observed in vivo [94].
To perform the immunostaining experiments, we utilized detergent-free conditions so that we are examining only ECM that is deposited extracellularly [94] and
not intracellular ECM molecules. We utilized this approach in our in vitro 3D cultures but also stained an in vivo tissue, the quail chorioallantoic membrane, in the
same manner [94]. This is key point because we have shown that extracellular deposition of vascular basement membrane matrix is markedly stimulated by pericyte
1 Molecular Control of Capillary Tube Morphogenesis and Maturation Through…
about a 50% decrease in pericyte coverage of vessels [16]. Of interest is that these
mice show primary defects in microvessel beds (where pericyte coverage is present), while larger vessels are much less affected. This microvascular deficiency
phenotype manifests particularly in the kidney and central nervous system which
strongly resembles that observed in diabetic microangiopathy [16]. Loss of pericytes is a major pathogenic cause of this type of microvascular disease [51]. It is
important to further understand the signaling mechanisms which underlie pericyte
recruitment to EC-lined tubes, to both identify other factors that regulate this
recruitment as well as understand how pericytes invade 3D matrices to recruit to
these tubes. This is currently a major research direction of our laboratory.
1.3.14 Pericyte Recruitment to EC-Lined Tubes Stimulates
ECM Remodeling Events and Vascular Basement
Membrane Matrix Assembly
Using our new model of EC-pericyte tube co-assembly, we sought to identify how
pericytes contribute to vascular tube maturation and stabilization events. At different time points of tube co-assembly, we performed transmission electron microscopy and immunofluorescence microscopy to examine if basement membrane
matrix assembly occurred [94]. In Fig. 1.5b, we show immunostaining for the EC
marker CD31, while the pericytes stably express green fluorescent protein (GFP).
This image shows EC tube networks that have associated pericytes at day 5 of culture. We also show a light microscopy image of the coculture system in Fig. 1.5a.
As shown in Fig. 1.5c, d, there is marked deposition of laminin and collagen type
IV, two critical basement membrane matrix components. In addition, we reported
that fibronectin, nidogen-1, nidogen-2, and perlecan were also deposited around
EC-lined tubes only when pericytes were cocultured with ECs [94] (Fig. 1.5e). We
demonstrated that basement membrane matrices were observed by transmission
electron microscopy, only when EC-pericytes were cocultured [94], and over many
years we have never observed basement membrane deposition in the absence of
pericytes in electron microscopic studies. Further, we confirmed our results in vivo
and demonstrated that pericyte recruitment to developing quail EC tubes directly
correlates with vascular basement membrane assembly, at day 7 of embryonic
development [94]. Prior to pericyte recruitment, no vascular basement membranes
around EC tubes were observed in vivo [94].
To perform the immunostaining experiments, we utilized detergent-free conditions so that we are examining only ECM that is deposited extracellularly [94] and
not intracellular ECM molecules. We utilized this approach in our in vitro 3D cultures but also stained an in vivo tissue, the quail chorioallantoic membrane, in the
same manner [94]. This is key point because we have shown that extracellular deposition of vascular basement membrane matrix is markedly stimulated by pericyte
1 Molecular Control of Capillary Tube Morphogenesis and Maturation Through…