In pathological scenarios, such as tumor growth and diabetic retinopathy, blocking angiogenesis would be beneficial. In others, such as myocardial infarction and hypertension, promoting angiogenesis might be desirable. Due to their putative influence on endothelial cells, vascular pericytes have become a topic of growing interest and are increasingly being evaluated as a potential target for angioregulatory therapies. For example, the strategy of manipulating pericyte recruitment to capillaries could result in anti- or pro-angiogenic effects. However, our current understanding of pericytes is limited by knowledge gaps regarding pericyte identity and lineage. To use a music analogy, this review is a “mash-up” that attempts to integrate what we know about pericyte functionality and expression with what is beginning to be elucidated regarding their regenerative potential. We explore the lingering questions regarding pericyte phenotypic identity and lineage. The expression of different pericyte markers (e.g., SMA, Desmin, NG2 and PDGFR-β) varies for different subpopulations and tissues. Previous use of these markers to identify pericytes has suggested potential phenotypic overlaps and plasticity toward other cell phenotypes. Our review chronicles the state of the literature, identifies critical unanswered questions, and motivates future research aimed at understanding this intriguing cell type and harnessing its therapeutic potential.

Objective Chronic arterial occlusion results in arteriogenesis of collateral blood vessels. This process has been shown to be dependent upon the recruitment of growth-promoting macrophages to remodeling collaterals. However, the potential role of venules in monocyte recruitment during microvascular arteriogenesis is not well demonstrated. First, we aim to document that arteriogenesis occurs in the mouse spinotrapezius ligation model. Then, we investigate the temporal and spatial distribution, as well as proliferation, of monocytes/macrophages recruited to collateral arterioles in response to elevated fluid shear stress. Approach and Results Laser speckle flowmetry confirmed a post-ligation increase in blood velocity within collateral arterioles but not venules. After 72 hours post-ligation, collateral arteriole diameters were increased, proliferating cells were identified in vessel walls of shear-activated collaterals, and perivascular CD206+ macrophages demonstrated proliferation. An EdU assay identified proliferation. CD68+CD206+ cells around collaterals were increased 96%, while CX3CR1(+/GFP ) cells were increased 126% in ligated versus sham groups after 72 hours. CX3CR1(+/GFP ) cells were predominately venule-associated at 6 hours post-ligation; and CX3CR1(+/GFP hi) cells shifted from venule-associated to arteriole-associated between 6 and 72 hours post-surgery exclusively in ligated muscle. We report accumulation and extravasation of adhered CX3CR1(+/GFP) cells in and from venules, but not arterioles, following ligation. Conclusions Our results demonstrate that arteriogenesis occurs in the murine spinotrapezius ligation model and implicate post-capillary venules as the site of tissue entry for circulating monocytes. Local proliferation of macrophages also is documented. These data open up questions concerning the role of arteriole-venule communication during monocyte recruitment.

Diabetic retinopathy is characterized by progressive vascular dropout with subsequent vision loss. We have recently shown that an intravitreal injection of adipose-derived stem cells (ASCs) can stabilize the retinal microvasculature, enabling repair and regeneration of damaged capillary beds in vivo. Because an understanding of ASC status from healthy versus diseased donors will be important as autologous cellular therapies are developed for unmet clinical needs, we took advantage of the hyperglycemic Akimba mouse as a preclinical in vivo model of diabetic retinopathy in an effort aimed at evaluating therapeutic efficacy of adipose-derived stem cells (mASCs) derived either from healthy, nondiabetic or from diabetic mice. To these ends, Akimba mice received intravitreal injections of media conditioned by mASCs or mASCs themselves, subsequent to development of substantial retinal capillary dropout. mASCs from healthy mice were more effective than diabetic mASCs in protecting the diabetic retina from further vascular dropout. Engrafted ASCs were found to preferentially associate with the retinal vasculature. Conditioned medium was unable to recapitulate the vasoprotection seen with injected ASCs. In vitro diabetic ASCs showed decreased proliferation and increased apoptosis compared with healthy mASCs. Diabetic ASCs also secreted less vasoprotective factors than healthy mASCs, as determined by high-throughput enzymelinked immunosorbent assay. Our findings suggest that diabetic ASCs are functionally impaired compared with healthy ASCs and support the utility of an allogeneic injection of ASCs versus autologous or conditioned media approaches in the treatment of diabetic retinopathy. STEM CELLS TRANSLATIONAL MEDICINE 2015;4:459-467 SIGNIFICANCEThis work addresses the therapeutic potential of stem cells that are obtained from diabetic donors, and the clinical focus of our work is diabetic retinopathy. This work examined how injected murine adiposederived stem cells (ASCs) affect the retinal microvasculature in a mouse model of diabetic retinopathy. Results show that ASCs obtained from healthy mice secrete angiogenic growth factors and promote retinal vascular stability when they are injected intravitreally. The findings further suggest that ASCs obtained from diabetic mice have a diminished ability to support the retinal vasculature in this mouse model of retinal vasculopathy.

Diabetic retinopathy is a potentially blinding eye disease that threatens the vision of one-ninth of patients with diabetes. Progression of the disease has long been attributed to an initial dropout of pericytes that enwrap the retinal microvasculature. Revealed through retinal vascular digests, a subsequent increase in basement membrane bridges was also observed. Using cell-specific markers, we demonstrate that pericytes rather than endothelial cells colocalize with these bridges. We show that the density of bridges transiently increases with elevation of Ang-2, PDGF-BB, and blood glucose; is rapidly reversed on a timescale of days; and is often associated with a pericyte cell body located off vessel. Cell-specific knockout of KLF4 in pericytes fully replicates this phenotype. In vivo imaging of limbal vessels demonstrates pericyte migration off vessel, with rapid pericyte filopodial-like process formation between adjacent vessels. Accounting for off-vessel and on-vessel pericytes, we observed no pericyte loss relative to nondiabetic control retina. These findings reveal the possibility that pericyte perturbations in location and process formation may play a role in the development of pathological vascular remodeling in diabetic retinopathy.

The stem cell pluripotency factor Oct4 serves a critical protective role during atherosclerotic plaque development by promoting smooth muscle cell (SMC) investment. Here, we show using Myh11-CreER T2 lineage-tracing with inducible SMC and pericyte (SMC-P) knockout of Oct4 that Oct4 regulates perivascular cell migration and recruitment during angiogenesis. Knockout of Oct4 in perivascular cells significantly impairs perivascular cell migration, increases perivascular cell death, delays endothelial cell migration, and promotes vascular leakage following corneal angiogenic stimulus. Knockout of Oct4 in perivascular cells also impairs perfusion recovery and decreases angiogenesis following hindlimb ischemia. Transcriptomic analyses demonstrate that expression of the migratory gene Slit3 is reduced following loss of Oct4 in cultured SMCs, and in Oct4-deficient perivascular cells in ischemic hindlimb muscle. Together, these results provide evidence that Oct4 plays an essential role within perivascular cells in injury- and hypoxia-induced angiogenesis.

Vascular endothelial growth factor receptor‐3 (VEGFR3) signalling promotes lymphangiogenesis. While there are many reported mechanisms of VEGFR3 activation, there is little understanding of how VEGFR3 signalling is attenuated to prevent lymphatic vascular overgrowth and ensure proper lymph vessel development. Here, we show that endothelial cell‐specific depletion of integrin‐linked kinase (ILK) in mouse embryos hyper‐activates VEGFR3 signalling and leads to overgrowth of the jugular lymph sacs/primordial thoracic ducts, oedema and embryonic lethality. Lymphatic endothelial cell (LEC)‐specific deletion of Ilk in adult mice initiates lymphatic vascular expansion in different organs, including cornea, skin and myocardium. Knockdown of ILK in human LECs triggers VEGFR3 tyrosine phosphorylation and proliferation. ILK is further found to impede interactions between VEGFR3 and β1 integrin in vitro and in vivo, and endothelial cell‐specific deletion of an Itgb1 allele rescues the excessive lymphatic vascular growth observed upon ILK depletion. Finally, mechanical stimulation disrupts the assembly of ILK and β1 integrin, releasing the integrin to enable its interaction with VEGFR3. Our data suggest that ILK facilitates mechanically regulated VEGFR3 signalling via controlling its interaction with β1 integrin and thus ensures proper development of lymphatic vessels.

Microvascular endothelial cell heterogeneity and its relationship to hemodynamics remains poorly understood due to a lack of sufficient methods to examine these parameters in vivo at high resolution throughout an angiogenic network. The availability of surrogate markers for functional vascular proteins, such as green fluorescent protein, enables expression in individual cells to be followed over time using confocal microscopy, while photoacoustic microscopy enables dynamic measurement of blood flow across the network with capillary-level resolution. We combined these two non-invasive imaging modalities in order to spatially and temporally analyze biochemical and biomechanical drivers of angiogenesis in murine corneal neovessels. By stimulating corneal angiogenesis with an alkali burn in Tie2-GFP fluorescent-reporter mice, we evaluated how onset of blood flow and surgically-altered blood flow affects Tie2-GFP expression. Our study establishes a novel platform for analyzing heterogeneous blood flow and fluorescent reporter protein expression across a dynamic microvascular network in an adult mammal.

Recent smooth muscle cell (SMC) lineage-tracing studies have revealed that SMCs undergo remarkable changes in phenotype during development of atherosclerosis. Of major interest, we demonstrated that Kruppel-like factor 4 (KLF4) in SMCs is detrimental for overall lesion pathogenesis, in that SMC-specific conditional knockout of the KLF4 gene ( Klf4) resulted in smaller, more-stable lesions that exhibited marked reductions in the numbers of SMC-derived macrophage- and mesenchymal stem cell-like cells. However, since the clinical consequences of atherosclerosis typically occur well after our reproductive years, we sought to identify beneficial KLF4-dependent SMC functions that were likely to be evolutionarily conserved. We tested the hypothesis that KLF4-dependent SMC transitions play an important role in the tissue injury-repair process. Using SMC-specific lineage-tracing mice positive and negative for simultaneous SMC-specific conditional knockout of Klf4, we demonstrate that SMCs in the remodeling heart after ischemia-reperfusion injury (IRI) express KLF4 and transition to a KLF4-dependent macrophage-like state and a KLF4-independent myofibroblast-like state. Moreover, heart failure after IRI was exacerbated in SMC Klf4 knockout mice. Surprisingly, we observed a significant cardiac dilation in SMC Klf4 knockout mice before IRI as well as a reduction in peripheral resistance. KLF4 chromatin immunoprecipitation-sequencing analysis on mesenteric vascular beds identified potential baseline SMC KLF4 target genes in numerous pathways, including PDGF and FGF. Moreover, microvascular tissue beds in SMC Klf4 knockout mice had gaps in lineage-traced SMC coverage along the resistance arteries and exhibited increased permeability. Together, these results provide novel evidence that Klf4 has a critical maintenance role within microvascular SMCs: it is required for normal SMC function and coverage of resistance arteries. NEW & NOTEWORTHY We report novel evidence that the Kruppel-like factor 4 gene ( Klf4) has a critical maintenance role within microvascular smooth muscle cells (SMCs). SMC-specific Klf4 knockout at baseline resulted in a loss of lineage-traced SMC coverage of resistance arteries, dilation of resistance arteries, increased blood flow, and cardiac dilation.