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Endothelial expression of TGFβ type II receptor is required to maintain vascular integrity during postnatal development of the central nervous system.

Allinson KR, Lee HS, Fruttiger M, McCarty JH, McCarty J, Arthur HM - PLoS ONE (2012)

Bottom Line: TGFβ signalling in endothelial cells is important for angiogenesis in early embryonic development, but little is known about its role in early postnatal life.Instead the leading endothelial cells formed glomerular tufts with associated smooth muscle cells.Thus, TGFβ signalling is essential in vascular endothelial cells for maintaining vascular integrity at the angiogenic front as it migrates into developing neural tissues in early postnatal life.

View Article: PubMed Central - PubMed

Affiliation: Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom.

ABSTRACT
TGFβ signalling in endothelial cells is important for angiogenesis in early embryonic development, but little is known about its role in early postnatal life. To address this we used a tamoxifen inducible Cre-LoxP strategy in neonatal mice to deplete the TypeII TGFβ receptor (Tgfbr2) specifically in endothelial cells. This resulted in multiple micro-haemorrhages, and glomeruloid-like vascular tufts throughout the cerebral cortices and hypothalamus of the brain as well as in retinal tissues. A detailed examination of the retinal defects in these mutants revealed that endothelial adherens and tight junctions were in place, pericytes were recruited and there was no failure of vascular smooth muscle differentiation. However, the deeper retinal plexus failed to form in these mutants and the angiogenic sprouts stalled in their progress towards the inner nuclear layer. Instead the leading endothelial cells formed glomerular tufts with associated smooth muscle cells. This evidence suggests that TGFβ signalling is not required for vessel maturation, but is essential for the organised migration of endothelial cells as they begin to enter the deeper layers of the retina. Thus, TGFβ signalling is essential in vascular endothelial cells for maintaining vascular integrity at the angiogenic front as it migrates into developing neural tissues in early postnatal life.

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Related in: MedlinePlus

Endothelial glomerular tufts in the Tgfbr2-iKOe mutants contain multiple smooth muscle cells.Immunofluorescent staining of P14 retinal paraffin sections with isolectin-alexa488 and anti-alpha smooth muscle actin (aSMA) conjugated to Cy3 show the typical non-muscularised microvessels of the control retinal plexus (arrows in A and D), whereas high numbers of vascular smooth muscle cells are associated with endothelial glomerular tufts (E,F and H). Scale bar: 50 µm.
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pone-0039336-g006: Endothelial glomerular tufts in the Tgfbr2-iKOe mutants contain multiple smooth muscle cells.Immunofluorescent staining of P14 retinal paraffin sections with isolectin-alexa488 and anti-alpha smooth muscle actin (aSMA) conjugated to Cy3 show the typical non-muscularised microvessels of the control retinal plexus (arrows in A and D), whereas high numbers of vascular smooth muscle cells are associated with endothelial glomerular tufts (E,F and H). Scale bar: 50 µm.

Mentions: The interaction of ECs with vascular smooth muscle cells/pericytes is critical during neural angiogenesis and TGFβ signalling from ECs has been reported to promote the maturation of vascular smooth muscle cells during vascular development [25]. Also, we have previously shown that defects in TGFβ signalling in ECs can lead to reduced vessel muscularisation in the yolk sac [26]. We therefore expected to see reduced muscularisation of vessels in Tgfbr2-iKOe mutants, However, we observed no reduction in smooth muscle cells (detected by staining for alpha smooth muscle actin) in mutants compared with controls (Figure S7A–D). Surprisingly, the numbers of vascular smooth muscle cells were actually increased in Tgfbr2-iKOe retinal capillaries compared with controls, a feature that was first observed at P7 (Figure S7E,F). Furthermore, the glomeruloid tufts also contained numerous vascular smooth muscle cells (Figure 6) that were proliferating in a similar way to the ECs (Figure S8). A 3D reconstruction of confocal z-slices illustrates the contrast between the non-muscularised capillary network of the primary and secondary plexus in the normal retina and the muscularised glomerular tufts that form as downward growths from the primary plexus penetrate the neural tissue (see movies S1 and S2). Thus, there was an abnormal distribution of proliferating vascular smooth muscle cells, but no evidence for a defect in smooth muscle cell differentiation. As pericytes are key to the generation of the BRB, we also used anti-desmin and anti-NG2 antibodies to examine the organisation of pericytes on developing retinal vessels. Surprisingly we observed no differences in pericyte organisation on retinal vasculature except that the endothelial glomerular tufts present in the mutants contained pericytes (Figure 7). Taken together this data suggests there was not a failure in vascular smooth muscle cell differentiation or recruitment in the absence of endothelial Tgfbr2, but rather there was a failure of progression of the angiogenic sprouts into the neural plexus resulting in an aggregate of proliferating and disorganised ECs with associated pericytes and vascular smooth muscle cells. In order to compare with another tissue undergoing active angiogenesis at the same period of development we examined the ear vasculature of mutants and controls. We confirmed that there were no abnormal glomerular tufts in this tissue in the absence of endothelial Tgfbr2 (Figure S9), suggesting this phenotype is specific for angiogenesis of neural tissues.


Endothelial expression of TGFβ type II receptor is required to maintain vascular integrity during postnatal development of the central nervous system.

Allinson KR, Lee HS, Fruttiger M, McCarty JH, McCarty J, Arthur HM - PLoS ONE (2012)

Endothelial glomerular tufts in the Tgfbr2-iKOe mutants contain multiple smooth muscle cells.Immunofluorescent staining of P14 retinal paraffin sections with isolectin-alexa488 and anti-alpha smooth muscle actin (aSMA) conjugated to Cy3 show the typical non-muscularised microvessels of the control retinal plexus (arrows in A and D), whereas high numbers of vascular smooth muscle cells are associated with endothelial glomerular tufts (E,F and H). Scale bar: 50 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3383742&req=5

pone-0039336-g006: Endothelial glomerular tufts in the Tgfbr2-iKOe mutants contain multiple smooth muscle cells.Immunofluorescent staining of P14 retinal paraffin sections with isolectin-alexa488 and anti-alpha smooth muscle actin (aSMA) conjugated to Cy3 show the typical non-muscularised microvessels of the control retinal plexus (arrows in A and D), whereas high numbers of vascular smooth muscle cells are associated with endothelial glomerular tufts (E,F and H). Scale bar: 50 µm.
Mentions: The interaction of ECs with vascular smooth muscle cells/pericytes is critical during neural angiogenesis and TGFβ signalling from ECs has been reported to promote the maturation of vascular smooth muscle cells during vascular development [25]. Also, we have previously shown that defects in TGFβ signalling in ECs can lead to reduced vessel muscularisation in the yolk sac [26]. We therefore expected to see reduced muscularisation of vessels in Tgfbr2-iKOe mutants, However, we observed no reduction in smooth muscle cells (detected by staining for alpha smooth muscle actin) in mutants compared with controls (Figure S7A–D). Surprisingly, the numbers of vascular smooth muscle cells were actually increased in Tgfbr2-iKOe retinal capillaries compared with controls, a feature that was first observed at P7 (Figure S7E,F). Furthermore, the glomeruloid tufts also contained numerous vascular smooth muscle cells (Figure 6) that were proliferating in a similar way to the ECs (Figure S8). A 3D reconstruction of confocal z-slices illustrates the contrast between the non-muscularised capillary network of the primary and secondary plexus in the normal retina and the muscularised glomerular tufts that form as downward growths from the primary plexus penetrate the neural tissue (see movies S1 and S2). Thus, there was an abnormal distribution of proliferating vascular smooth muscle cells, but no evidence for a defect in smooth muscle cell differentiation. As pericytes are key to the generation of the BRB, we also used anti-desmin and anti-NG2 antibodies to examine the organisation of pericytes on developing retinal vessels. Surprisingly we observed no differences in pericyte organisation on retinal vasculature except that the endothelial glomerular tufts present in the mutants contained pericytes (Figure 7). Taken together this data suggests there was not a failure in vascular smooth muscle cell differentiation or recruitment in the absence of endothelial Tgfbr2, but rather there was a failure of progression of the angiogenic sprouts into the neural plexus resulting in an aggregate of proliferating and disorganised ECs with associated pericytes and vascular smooth muscle cells. In order to compare with another tissue undergoing active angiogenesis at the same period of development we examined the ear vasculature of mutants and controls. We confirmed that there were no abnormal glomerular tufts in this tissue in the absence of endothelial Tgfbr2 (Figure S9), suggesting this phenotype is specific for angiogenesis of neural tissues.

Bottom Line: TGFβ signalling in endothelial cells is important for angiogenesis in early embryonic development, but little is known about its role in early postnatal life.Instead the leading endothelial cells formed glomerular tufts with associated smooth muscle cells.Thus, TGFβ signalling is essential in vascular endothelial cells for maintaining vascular integrity at the angiogenic front as it migrates into developing neural tissues in early postnatal life.

View Article: PubMed Central - PubMed

Affiliation: Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom.

ABSTRACT
TGFβ signalling in endothelial cells is important for angiogenesis in early embryonic development, but little is known about its role in early postnatal life. To address this we used a tamoxifen inducible Cre-LoxP strategy in neonatal mice to deplete the TypeII TGFβ receptor (Tgfbr2) specifically in endothelial cells. This resulted in multiple micro-haemorrhages, and glomeruloid-like vascular tufts throughout the cerebral cortices and hypothalamus of the brain as well as in retinal tissues. A detailed examination of the retinal defects in these mutants revealed that endothelial adherens and tight junctions were in place, pericytes were recruited and there was no failure of vascular smooth muscle differentiation. However, the deeper retinal plexus failed to form in these mutants and the angiogenic sprouts stalled in their progress towards the inner nuclear layer. Instead the leading endothelial cells formed glomerular tufts with associated smooth muscle cells. This evidence suggests that TGFβ signalling is not required for vessel maturation, but is essential for the organised migration of endothelial cells as they begin to enter the deeper layers of the retina. Thus, TGFβ signalling is essential in vascular endothelial cells for maintaining vascular integrity at the angiogenic front as it migrates into developing neural tissues in early postnatal life.

Show MeSH
Related in: MedlinePlus