Lymphatic endothelial cell RXRα is critical for 9-cis-retinoic acid-mediated lymphangiogenesis and prevention of secondary lymphedema.

Secondary lymphedema is a debilitating disease characterized by abnormal soft tissue swelling and caused by lymphatic system dysfunction. Despite a high prevalence of secondary lymphedema after cancer treatments, current management is supportive and there are no approved therapeutic agents that can thwart disease progression. We have previously demonstrated that 9-cis-retinoic acid (9-cisRA) has the potential to be repurposed for lymphedema as it mitigates disease by promoting lymphangiogenesis at the site of lymphatic injury. Although the efficacy of 9-cisRA has been demonstrated in previous studies, the mechanism of action is not completely understood. In this study, we demonstrate that when RXRα is specifically deleted in lymphatic endothelial cells, 9-cisRA fails to induce lymphangiogenesis in vitro and prevent pathologic progression of postsurgical lymphedema in vivo. These findings demonstrate that downstream nuclear receptor RXRα plays a critical role in the therapeutic efficacy of 9-cisRA in postsurgical lymphedema.

ORAI1 Activates Proliferation of Lymphatic Endothelial Cells in Response to Laminar Flow Through Krüppel-Like Factors 2 and 4.

RATIONALE: Lymphatic vessels function to drain interstitial fluid from a variety of tissues. Although shear stress generated by fluid flow is known to trigger lymphatic expansion and remodeling, the molecular basis underlying flow-induced lymphatic growth is unknown. OBJECTIVE: We aimed to gain a better understanding of the mechanism by which laminar shear stress activates lymphatic proliferation. METHODS AND RESULTS: Primary endothelial cells from dermal blood and lymphatic vessels (blood vascular endothelial cells and lymphatic endothelial cells [LECs]) were exposed to low-rate steady laminar flow. Shear stress-induced molecular and cellular responses were defined and verified using various mutant mouse models. Steady laminar flow induced the classic shear stress responses commonly in blood vascular endothelial cells and LECs. Surprisingly, however, only LECs showed enhanced cell proliferation by regulating the vascular endothelial growth factor (VEGF)-A, VEGF-C, FGFR3, and p57/CDKN1C genes. As an early signal mediator, ORAI1, a pore subunit of the calcium release-activated calcium channel, was identified to induce the shear stress phenotypes and cell proliferation in LECs responding to the fluid flow. Mechanistically, ORAI1 induced upregulation of Krüppel-like factor (KLF)-2 and KLF4 in the flow-activated LECs, and the 2 KLF proteins cooperate to regulate VEGF-A, VEGF-C, FGFR3, and p57 by binding to the regulatory regions of the genes. Consistently, freshly isolated LECs from Orai1 knockout embryos displayed reduced expression of KLF2, KLF4, VEGF-A, VEGF-C, and FGFR3 and elevated expression of p57. Accordingly, mouse embryos deficient in Orai1, Klf2, or Klf4 showed a significantly reduced lymphatic density and impaired lymphatic development. CONCLUSIONS: Our study identified a molecular mechanism for laminar flow-activated LEC proliferation.

Prevention of Postsurgical Lymphedema by 9-cis Retinoic Acid.

OBJECTIVE: To determine the effect of 9-cis retinoic acid (9-cis RA) on postsurgical lymphedema. BACKGROUND: 9-cis RA promotes lymphangiogenesis in vitro and in vivo and has promise as a therapeutic agent to limit the development of postsurgical lymphedema. METHODS: Lymphedema was induced in the right hind limb after a single fraction of 20 Gy radiation, popliteal lymphadenectomy, and lymphatic vessel ablation. Postoperatively, mice were randomly divided in to 2 groups that received daily intraperitoneal injections of either (1) an oil-based vehicle solution (control) or (2) 0.08 mg/kg of 9-cis RA dissolved in a vehicle solution. Outcome measures included paw thickness, lymphatic drainage, and lymphatic vessel density as measured by podoplanin immunohistochemistry and whole mount skin analysis. RESULTS: Using our combined injury protocol, postsurgical lymphedema was observed 89% of the time. 9-cis RA-treated animals had less early postsurgical edema and significantly less paw lymphedema compared with vehicle-treated animals at all time-points (P < 0.001). 9-cis RA-treated animals had significantly faster lymphatic drainage as measured by indocyanine green clearance and increased lymphatic vessel density as measured by podoplanin immunohistochemistry (P < 0.001) and whole mount skin analysis (P < 0.05). CONCLUSIONS: We have developed a highly reproducible model of secondary lymphedema and have demonstrated that 9-cis RA significantly prevents postsurgical lymphedema. Treatment with 9-cis RA is associated with increased lymphatic clearance and lymphangiogenesis. Because 9-cis RA (alitretinoin) is already approved for clinical use by the US Food and Drug Administration for other conditions, it has the potential to be repurposed as a preventative agent for postsurgical lymphedema in humans.

CXCR2 inhibition enhances sulindac-mediated suppression of colon cancer development.

Small chemical compound sulindac has been approved as a preventive approach against colon cancer for its effectiveness in treatment of precancerous adenoma. Due to its severe toxicities in the cardiovascular, gastrointestinal and renal systems, however, a combination of low-dose sulindac with other chemopreventive agents has been sought after as an alternative therapeutic strategy that could increase its effectiveness, while minimizing its adverse effects. To identify the promising alternative approach, we investigated the therapeutic potential of targeting the interleukin (IL)-8/CXCR2 pathway in colon cancer treatment using both loss-of-function (CXCR2 knockout) and gain-of-function (IL-8 overexpression) mouse models, as the IL-8/CXCR2 pathway has been shown to be activated in intestinal tumors of both human and experimental animals. We found that deletion of CXCR2 gene and ectopic expression of IL-8 suppresses and enhances, respectively, intestinal tumor development caused by a mutation in the APC gene. Moreover, a single copy deletion of CXCR2 gene resulted in abrogation of COX-2 and Gro-α upregulation in intestinal tumors caused by the APC mutation. Moreover, a single copy (heterozygote) deletion of CXCR2 gene was sufficient to synergize with a low-dose sulindac treatment in suppressing APCmin-induced intestinal polyposis. Together, our study provides a therapeutic justification of combined inhibition of CXCR2 and sulindac treatment in colon cancer prevention.

Prolymphangiogenic Effects of 9-cis Retinoic Acid Are Enhanced at Sites of Lymphatic Injury and Dependent on Treatment Duration in Experimental Postsurgical Lymphedema.

Background: Patients undergoing surgical treatment for solid tumors are at risk for development of secondary lymphedema due to intraoperative lymphatic vessel injury. The damaged lymphatic vessels fail to adequately regenerate and lymphatic obstruction leads to fluid and protein accumulation in the interstitial space and chronic lymphedema develops as a result. There are currently no effective pharmacological agents that reduce the risk of developing lymphedema or treat pre-existing lymphedema, and management is largely palliative. The present study investigated the efficacy of various 9-cis retinoic acid (9-cis RA) dosing strategies in reducing postsurgical lymphedema by utilizing a well-established mouse tail lymphedema model. Methods and Results: Short-duration treatment with 9-cis RA did not demonstrate a significant reduction in postoperative tail volume, nor an improvement in lymphatic clearance. However, long-term treatment with 9-cis RA resulted in decreased overall tail volume, dermal thickness, and epidermal thickness, with an associated increase in functional lymphatic clearance and lymphatic vessel density, assessed by LYVE-1 immunostaining, compared with control. These effects were seen at the site of lymphatic injury, with no significant changes observed in uninjured sites such as ear skin and the diaphragm. Conclusions: Given the reported results indicating that 9-cis RA is a potent promoter of lymphangiogenesis and improved lymphatic clearance at sites of lymphatic injury, investigation of postoperative 9-cis RA administration to patients at high risk of developing lymphedema may demonstrate positive efficacy and reduced rates of postsurgical lymphedema.

The Lymphatic Cell Environment Promotes Kaposi Sarcoma Development by Prox1-Enhanced Productive Lytic Replication of Kaposi Sarcoma Herpes Virus.

Kaposi sarcoma is the most common cancer in human immunodeficiency virus-positive individuals and is caused by Kaposi sarcoma-associated herpesvirus (KSHV). It is believed that a small number of latently infected Kaposi sarcoma tumor cells undergo spontaneous lytic reactivation to produce viral progeny for infection of new cells. Here, we use matched donor-derived human dermal blood and lymphatic endothelial cells (BEC and LEC, respectively) to show that KSHV-infected BECs progressively lose viral genome as they proliferate. In sharp contrast, KSHV-infected LECs predominantly entered lytic replication, underwent cell lysis, and released new virus. Continuous lytic cell lysis and de novo infection allowed LEC culture to remain infected for a prolonged time. Because of the strong propensity of LECs toward lytic replication, LECs maintained virus as a population, despite the death of individual host cells from lytic lysis. The master regulator of lymphatic development, Prox1, bound the promoter of the RTA gene to upregulate its expression and physically interacted with RTA protein to coregulate lytic genes. Thus, LECs may serve as a proficient viral reservoir that provides viral progeny for continuous de novo infection of tumor origin cells, and potentially BECs and mesenchymal stem cells, which give rise to Kaposi sarcoma tumors. Our study reveals drastically different host cell behaviors between BEC and LEC and defines the underlying mechanisms of the lymphatic cell environment supporting persistent infection in Kaposi sarcoma tumors. SIGNIFICANCE: This study defines the mechanism by which Kaposi's sarcoma could be maintained by virus constantly produced by lymphatic cells in HIV-positive individuals.

Organogenesis and distribution of the ocular lymphatic vessels in the anterior eye.

Glaucoma surgeries, such as trabeculectomy, are performed to lower intraocular pressure to reduce risk of vision loss. These surgeries create a new passage in the eye that reroutes the aqueous humor outflow to the subconjunctival space, where the fluid is presumably absorbed by the conjunctival lymphatics. Here, we characterized the development and function of the ocular lymphatics using transgenic lymphatic reporter mice and rats. We found that the limbal and conjunctival lymphatic networks are progressively formed from a primary lymphatic vessel that grows from the nasal-side medial canthus region at birth. This primary lymphatic vessel immediately branches out, invades the limbus and conjunctiva, and bidirectionally encircles the cornea. As a result, the distribution of the ocular lymphatics is significantly polarized toward the nasal side, and the limbal lymphatics are directly connected to the conjunctival lymphatics. New lymphatic sprouts are produced mainly from the nasal-side limbal lymphatics, posing the nasal side of the eye as more responsive to fluid drainage and inflammatory stimuli. Consistent with this polarized distribution of the ocular lymphatics, a higher drainage efficiency was observed in the nasal side than the temporal side of the eye when injected with a fluorescent tracer. In contrast, blood vessels are evenly distributed at the anterior surface of the eyes. Also, we found that these distinct vascular distribution patterns were conserved in human eyes. Together, our study demonstrated that the ocular surface lymphatics are more densely present in the nasal side and uncovered the potential clinical benefits in selecting the nasal side as a glaucoma surgery site to improve fluid drainage.

Mesenchymal Stromal Cells Isolated from Irradiated Human Skin Have Diminished Capacity for Proliferation, Differentiation, Colony Formation, and Paracrine Stimulation.

Ionizing radiation, commonly used in the treatment of solid tumors, has unintended but deleterious effects on overlying skin and is associated with chronic nonhealing wounds. Skin-derived mesenchymal stromal cells (SMSCs) are a pluripotent population of cells that are critically involved in skin homeostasis and wound healing. The aim of this study was to isolate and functionally characterize SMSCs from human skin that was previously irradiated as part of neoadjuvant or adjuvant cancer therapy. To this end, SMSCs were isolated from paired irradiated and nonirradiated human skin samples. Irradiated SMSCs expressed characteristic SMSC markers at lower levels, had disorganized cytoskeletal structure, and had disordered morphology. Functionally, these cells had diminished proliferative capacity and substantial defects in colony-forming capacity and differentiation in vitro. These changes were associated with significant differential expression of genes known to be involved in skin physiology and wound healing. Conditioned media obtained from irradiated SMSCs affected fibroblast but not endothelial cell proliferation and migration. These results suggest that in situ damage to SMSCs during neoadjuvant or adjuvant radiation may play a critical role in the pathogenesis of slow or nonhealing radiation wounds. Stem Cells Translational Medicine 2019;8:925&934.

Small Peptide Modulation of Fibroblast Growth Factor Receptor 3-Dependent Postnatal Lymphangiogenesis.

BACKGROUND: The fibroblast growth factor receptor (FGFR) family includes transmembrane receptors involved in a wide range of developmental and postdevelopmental biologic processes as well as a wide range of human diseases. In particular, FGFR3 has been implicated in the mechanism by which 9-cis retinoic acid (9-cisRA) induces lymphangiogenesis and improves lymphedema. The purpose of this study was to validate the efficacy of a novel small peptide FGFR3 inhibitor, peptide P3 (VSPPLTLGQLLS), and to elucidate the role of FGFR3 in 9-cisRA-induced lymphangiogenesis using this peptide. METHODS AND RESULTS: Peptide P3 effectively inhibited FGFR3 phosphorylation. In vitro, peptide P3-mediated FGFR3 inhibition did not decrease lymphatic endothelial cell (LEC) proliferation, migration, or tubule formation. However, peptide P3-mediated FGFR3 inhibition did block 9-cisRA-stimulated LEC proliferation, migration, and tubule formation. In vivo, peptide P3-mediated FGFR3 inhibition was sufficient to inhibit 9-cisRA-induced tracheal lymphangiogenesis. CONCLUSION: FGFR3 does not appear to be essential to nonpromoted LEC proliferation, migration, and tubule formation. However, FGFR3 may play a key role in LEC proliferation, migration, tubule formation, and postnatal in vivo lymphangiogenesis when pharmacologically induced by 9-cisRA. P3 may have the potential to be used as a precise regulatory control element for 9-cisRA-mediated lymphangiogenesis.

Piezo1 incorporates mechanical force signals into the genetic program that governs lymphatic valve development and maintenance.

The lymphatic system plays crucial roles in tissue homeostasis, lipid absorption, and immune cell trafficking. Although lymphatic valves ensure unidirectional lymph flows, the flow itself controls lymphatic valve formation. Here, we demonstrate that a mechanically activated ion channel Piezo1 senses oscillating shear stress (OSS) and incorporates the signal into the genetic program controlling lymphatic valve development and maintenance. Time-controlled deletion of Piezo1 using a pan-endothelial Cre driver (Cdh5[PAC]-CreERT2) or lymphatic-specific Cre driver (Prox1-CreERT2) equally inhibited lymphatic valve formation in newborn mice. Furthermore, Piezo1 deletion in adult lymphatics caused substantial lymphatic valve degeneration. Piezo1 knockdown in cultured lymphatic endothelial cells (LECs) largely abrogated the OSS-induced upregulation of the lymphatic valve signature genes. Conversely, ectopic Piezo1 overexpression upregulated the lymphatic valve genes in the absence of OSS. Remarkably, activation of Piezo1 using chemical agonist Yoda1 not only accelerated lymphatic valve formation in animals, but also triggered upregulation of some lymphatic valve genes in cultured LECs without exposure to OSS. In summary, our studies together demonstrate that Piezo1 is the force sensor in the mechanotransduction pathway controlling lymphatic valve development and maintenance, and Piezo1 activation is a potentially novel therapeutic strategy for congenital and surgery-associated lymphedema.

Human Acellular Dermis as Spacer for Small-Joint Arthroplasty: Analysis of Revascularization in a Rabbit Trapeziectomy Model.

BACKGROUND: Carpometacarpal joint osteoarthritis affects 8 to 12 percent of the general population. Surgical management provides symptomatic relief for 78 percent of patients who fail conservative therapy, but little consensus exists regarding which surgical procedure provides superior patient outcomes. Recent human trials substituted exogenous acellular dermal matrices in the bone space, but there are no quantitative histologic data on the outcome of acellular dermal matrices in this environment. The authors aimed to quantify the revascularization and recellularization of acellular dermal matrices in the joint space using a rabbit model. METHODS: Bilateral lunate carpal bones were surgically removed in New Zealand rabbits. Acellular dermal matrix and autologous tissue were implanted in place of the lunate of the right and left wrists, respectively. Acellular dermal matrix was also implanted subcutaneously as a nonjoint control. Histologic and immunofluorescence analysis was performed after collection at 0, 6, and 12 weeks. RESULTS: Quantitative analysis of anti-α-smooth muscle actin and CD31 immunofluorescence revealed a sequential and comparable increase of vascular lumens in joint space and subcutaneous acellular dermal matrices. In contrast, autologous tissue implanted in the joint space did not have a similar increase in α-smooth muscle actin-positive or CD31-positive lumens. Semiquantitative analysis revealed increased cellularity in both autologous and acellular dermal matrix wrist implants at each time point, whereas average cellularity of subcutaneous acellular dermal matrix peaked at 6 weeks and regressed by 12 weeks. Trichrome and Sirius red staining revealed abundant collagen at all time points. CONCLUSION: The trapeziectomy joint space supports both cellular and vascular ingrowth into human acellular dermal matrix.

Laminar flow downregulates Notch activity to promote lymphatic sprouting.

The major function of the lymphatic system is to drain interstitial fluid from tissue. Functional drainage causes increased fluid flow that triggers lymphatic expansion, which is conceptually similar to hypoxia-triggered angiogenesis. Here, we have identified a mechanotransduction pathway that translates laminar flow-induced shear stress to activation of lymphatic sprouting. While low-rate laminar flow commonly induces the classic shear stress responses in blood endothelial cells and lymphatic endothelial cells (LECs), only LECs display reduced Notch activity and increased sprouting capacity. In response to flow, the plasma membrane calcium channel ORAI1 mediates calcium influx in LECs and activates calmodulin to facilitate a physical interaction between Krüppel-like factor 2 (KLF2), the major regulator of shear responses, and PROX1, the master regulator of lymphatic development. The PROX1/KLF2 complex upregulates the expression of DTX1 and DTX3L. DTX1 and DTX3L, functioning as a heterodimeric Notch E3 ligase, concertedly downregulate NOTCH1 activity and enhance lymphatic sprouting. Notably, overexpression of the calcium reporter GCaMP3 unexpectedly inhibited lymphatic sprouting, presumably by disturbing calcium signaling. Endothelial-specific knockouts of Orai1 and Klf2 also markedly impaired lymphatic sprouting. Moreover, Dtx3l loss of function led to defective lymphatic sprouting, while Dtx3l gain of function rescued impaired sprouting in Orai1 KO embryos. Together, the data reveal a molecular mechanism underlying laminar flow-induced lymphatic sprouting.

Development and Characterization of A Novel Prox1-EGFP Lymphatic and Schlemm's Canal Reporter Rat.

The lymphatic system plays a key role in tissue fluid homeostasis, immune cell trafficking, and fat absorption. We previously reported a bacterial artificial chromosome (BAC)-based lymphatic reporter mouse, where EGFP is expressed under the regulation of the Prox1 promoter. This reporter line has been widely used to conveniently visualize lymphatic vessels and other Prox1-expressing tissues such as Schlemm's canal. However, mice have a number of experimental limitations due to small body size. By comparison, laboratory rats are larger in size and more closely model the metabolic, physiological, and surgical aspects of humans. Here, we report development of a novel lymphatic reporter rat using the mouse Prox1-EGFP BAC. Despite the species mismatch, the mouse Prox1-EGFP BAC enabled a reliable expression of EGFP in Prox1-expressing cells of the transgenic rats and allowed a convenient visualization of all lymphatic vessels, including those in the central nervous system, and Schlemm's canal. To demonstrate the utility of this new reporter rat, we studied the contractile properties and valvular functions of mesenteric lymphatics, developed a surgical model for vascularized lymph node transplantation, and confirmed Prox1 expression in venous valves. Together, Prox1-EGFP rat model will contribute to the advancement of lymphatic research as a valuable experimental resource.

Efficient Assessment of Developmental, Surgical and Pathological Lymphangiogenesis Using a Lymphatic Reporter Mouse and Its Embryonic Stem Cells.

Several lymphatic reporter mouse lines have recently been developed to significantly improve imaging of lymphatic vessels. Nonetheless, the usage of direct visualization of lymphatic vessels has not been fully explored and documented. Here, we characterized a new Prox1-tdTomato transgenic lymphatic reporter mouse line, and demonstrated how this animal tool enables the researchers to efficiently assess developmental, surgical and pathological lymphangiogenesis by direct visualization of lymphatic vessels. Moreover, we have derived embryonic stem cells from this reporter line, and successfully differentiated them into lymphatic vessels in vivo. In conclusion, these experimental tools and techniques will help advance lymphatic research.

Aberrant Activation of Notch Signaling Inhibits PROX1 Activity to Enhance the Malignant Behavior of Thyroid Cancer Cells.

Papillary thyroid cancer (PTC) is one of the most common endocrine malignancies associated with significant morbidity and mortality. Although multiple studies have contributed to a better understanding of the genetic alterations underlying this frequently arising disease, the downstream molecular effectors that impact PTC pathogenesis remain to be further defined. Here, we report that the regulator of cell fate specification, PROX1, becomes inactivated in PTC through mRNA downregulation and cytoplasmic mislocalization. Expression studies in clinical specimens revealed that aberrantly activated NOTCH signaling promoted PROX1 downregulation and that cytoplasmic mislocalization significantly altered PROX1 protein stability. Importantly, restoration of PROX1 activity in thyroid carcinoma cells revealed that PROX1 not only enhanced Wnt/ß-catenin signaling but also regulated several genes known to be associated with PTC, including thyroid cancer protein (TC)-1, SERPINA1, and FABP4. Furthermore, PROX1 reexpression suppressed the malignant phenotypes of thyroid carcinoma cells, such as proliferation, motility, adhesion, invasion, anchorage-independent growth, and polyploidy. Moreover, animal xenograft studies demonstrated that restoration of PROX1 severely impeded tumor formation and suppressed the invasiveness and the nuclear/cytoplasmic ratio of PTC cells. Taken together, our findings demonstrate that NOTCH-induced PROX1 inactivation significantly promotes the malignant behavior of thyroid carcinoma and suggest that PROX1 reactivation may represent a potential therapeutic strategy to attenuate disease progression.