Corneal donor tissue preparation for endothelial keratoplasty

Over the past ten years, corneal transplantation surgical techniques have undergone revolutionary changes. Since its inception, traditional full thickness corneal transplantation has been the treatment to restore sight in those limited by corneal disease. Some disadvantages to this approach include a high degree of post-operative astigmatism, lack of predictable refractive outcome, and disturbance to the ocular surface. The development of Descemet's stripping endothelial keratoplasty (DSEK), transplanting only the posterior corneal stroma, Descemet's membrane, and endothelium, has dramatically changed treatment of corneal endothelial disease. DSEK is performed through a smaller incision; this technique avoids 'open sky' surgery with its risk of hemorrhage or expulsion, decreases the incidence of postoperative wound dehiscence, reduces unpredictable refractive outcomes, and may decrease the rate of transplant rejection. Initially, cornea donor posterior lamellar dissection for DSEK was performed manually resulting in variable graft thickness and damage to the delicate corneal endothelial tissue during tissue processing. Automated lamellar dissection (Descemet's stripping automated endothelial keratoplasty, DSAEK) was developed to address these issues. Automated dissection utilizes the same technology as LASIK corneal flap creation with a mechanical microkeratome blade that helps to create uniform and thin tissue grafts for DSAEK surgery with minimal corneal endothelial cell loss in tissue processing. Eye banks have been providing full thickness corneas for surgical transplantation for many years. In 2006, eye banks began to develop methodologies for supplying precut corneal tissue for endothelial keratoplasty. With the input of corneal surgeons, eye banks have developed thorough protocols to safely and effectively prepare posterior lamellar tissue for DSAEK surgery. This can be performed preoperatively at the eye bank. Research shows no significant difference in terms of the quality of the tissue or patient outcomes using eye bank precut tissue versus surgeon-prepared tissue for DSAEK surgery. For most corneal surgeons, the availability of precut DSAEK corneal tissue saves time and money, and reduces the stress of performing the donor corneal dissection in the operating room. In part because of the ability of the eye banks to provide high quality posterior lamellar corneal in a timely manner, DSAEK has become the standard of care for surgical management of corneal endothelial disease. The procedure that we are describing is the preparation of the posterior lamellar cornea at the eye bank for transplantation in DSAEK surgery (Figure 1).     

Validation of an endothelial roll preparation for Descemet Membrane Endothelial Keratoplasty by a cornea bank using “no touch” dissection technique

Descemet Membrane Endothelial Keratoplasty (DMEK) selectively replaces the damaged posterior part of the cornea. However, the DMEK technique relies on a manually-performed dissection that is time-consuming, requires training and presents a potential risk of endothelial graft damages leading to surgery postponement when performed by surgeons in the operative room. To validate precut corneal tissue preparation for DMEK provided by a cornea bank in order to supply a quality and security precut endothelial tissue. The protocol was a technology transfer from the Netherlands Institute for Innovative Ocular Surgery (NIIOS) to Lyon Cornea Bank, after formation in NIIOS to the DMEK “no touch” dissection technique. The technique has been validated in selected conditions (materials, microscope) and after a learning curve, cornea bank technicians prepared endothelial tissue for DMEK. Endothelial cells densities (ECD) were evaluated before and after preparation, after storage and transport to the surgery room. Microbiological and histological controls have been done. Twenty corneas were manually dissected; 18 without tears. Nineteen endothelial grafts formed a double roll. The ECD loss after cutting was 3.3 % (n = 19). After transportation 7 days later, we found an ECD loss of 25 % (n = 12). Three days after cutting and transportation, we found 2.1 % of ECD loss (n = 7). Histology found an endothelial cells monolayer lying on Descemet membrane. The mean thickness was 12 ± 2.2 µm (n = 4). No microbial contamination was found (n = 19). Endothelial roll stability has been validated at 3 days in our cornea bank. Cornea bank technicians trained can deliver to surgeons an ECD controlled, safety and ready to use endothelial tissue, for DMEK by “no touch” technique, allowing time saving, quality and security for surgeons.     

High Throughput Gene Expression Analysis Identifies Reliable Expression Markers of Human Corneal Endothelial Cells

Considerable interest has been generated for the development of suitable corneal endothelial graft alternatives through cell-tissue engineering, which can potentially alleviate the shortage of corneal transplant material. The advent of less invasive suture-less key-hole surgery options such as Descemet’s Stripping Endothelial Keratoplasty (DSEK) and Descemet’s Membrane Endothelial Keratoplasty (DMEK), which involve transplantation of solely the endothelial layer instead of full thickness cornea, provide further impetus for the development of alternative endothelial grafts for clinical applications. A major challenge for this endeavor is the lack of specific markers for this cell type. To identify genes that reliably mark corneal endothelial cells (CECs) in vivo and in vitro, we performed RNA-sequencing on freshly isolated human CECs (from both young and old donors), CEC cultures, and corneal stroma. Gene expression of these corneal cell types was also compared to that of other human tissue types. Based on high throughput comparative gene expression analysis, we identified a panel of markers that are: i) highly expressed in CECs from both young donors and old donors; ii) expressed in CECs in vivo and in vitro; and iii) not expressed in corneal stroma keratocytes and the activated corneal stroma fibroblasts. These were SLC4A11, COL8A2 and CYYR1. The use of this panel of genes in combination reliably ascertains the identity of the CEC cell type.     

Preparing Uniform-Thickness Corneal Endothelial Grafts from Donor Tissues Using a Non-Amplified Femtosecond Laser

Corneal grafts for Descemet’s Stripping Automated Endothelial Keratoplasty are commonly prepared using mechanical microkeratomes. However, the cuts produced in such way render corneal lenticules that are thinner centrally than peripherally, thus inducing a hyperopic shift. Here we describe a novel device for preparing donor corneal grafts, in which a single low-energy femtosecond laser system is used as both a light source for optical coherence tomography and for cutting the graft illuminating from the endothelial side. The same laser is first utilized to obtain three-dimensional optical coherence tomography images of the donor tissue for guiding the dissection and obtaining grafts of uniform thickness with no applanation or contact. This device allows an optimal procedure for preparing consistently thin posterior grafts for transplantation.     

Comparison of organ cultured precut corneas versus surgeon-cut corneas for Descemet’s stripping automated endothelial keratoplasty

To compare precut and surgeon-cut organ cultured donor corneas for DSAEK. A total of 119 consecutive eyes treated with DSAEK were retrospectically identified. 65 grafts were cut by the surgeon (Moria, ALTK System) prior to DSAEK and 54 grafts were precut by laboratory technicians from the Danish Eye Bank (Horizon single-use system). 1 year after surgery, tomographic images were obtained with the Pentacam HR. Endothelial cell density (ECD) and best-corrected visual acuity (BCVA) was determined. Graft thickness and graft asymmetry was evaluated in the centre and 1 mm from the edge of the graft in 6 semi-meridians. 1 year after surgery, the ECD loss was similar in the two groups, averaging 25.9 ± 14 % in surgeon-cut, and 22.9 ± 17 % in precut group (p = 0.33). Mean central graft thickness was 172 ± 6 μm in surgeon-cut grafts and 182 ± 6 μm in precut grafts (p = 0.30). BCVA was similar in surgeon-cut and precut corneas; being 0.25 ± 0.02 logMAR and 0.24 ± 0.02 logMAR, respectively (p = 0.59). The graft asymmetry index was 1.48 ± 0.02 for surgeon-cut and 1.44 ± 0.02 for precut grafts. There were no significant differences in complications rate in both groups. No correlations between BCVA and central graft thickness or graft asymmetry index in both groups were observed. Organ cultured precut donor corneas are comparable with surgeon-cut grafts with respect to ECD, graft thickness and asymmetry, and postoperative complication rate.     

Tectonic epikeratoplasty with ethanol-stored donor corneas

To evaluate the efficacy and outcomes of tectonic epikeratoplasty with use of ethanol-preserved corneal grafts for the management of perforated corneal melts. The present retrospective case series includes 10 eyes which underwent tectonic epikeratoplasty for perforated corneal melts. The stromal remainders of Descemet membrane endothelial keratoplasty (DMEK) and Descemet stripping automated endothelial keratoplasty (DSAEK) graft preparation were stored in 95% ethanol and used as emergency tectonic grafts for restoring globe integrity after sterile and infectious perforated corneal melts. In 6 cases with subtotal corneal melt, DMEK remainders (endothelium-denuded corneoscleral buttons) were used for ‘limbus to limbus’ tectonic epikeratoplasty and in 4 cases DSAEK remainders (anterior stroma) were used to seal focal perforated melts. Graft storage time was 5.1?±?4.9 (ranging from 0.5 to 17) months. The surgeries were successful in all cases with restitution of the globe integrity. During the postoperative course 4 cases developed a graft melt (corneoscleral button for limbus to limbus tectonic epikeratoplasty, n?=?3; lamellar patch, n?=?1) within 2–6 months after the initial procedure. Three patients underwent successful repeat tectonic epikeratoplasty. In the fourth case of graft melt the globe was enucleated due to underlying expulsive haemorrhage and severe pain. The short-term results of the present case series suggest that the use of ethanol-stored stromal remainder of donor corneas after endothelial keratoplasty is an efficient temporary measure for tectonic restoration of perforated corneas.     

PACAP through EGFR transactivation preserves human corneal endothelial integrity

The corneal endothelium is composed of a single hexagonal-shaped cells layer adherent to the Descemet's membrane. The primary function of these cells is maintaining of tissue clarity by regulating its hydration. Trauma, aging or other pathologies cause their loss, counterbalanced by enlargement of survived cells unable to guarantee an efficient fluid pumping to and from the stroma. Regenerative medicine using human corneal endothelial cells (HCECs) isolated from peripheral corneal-scleral tissue of a donor could be an attractive solution, overcoming transplantation problems. In a previous study, we have demonstrated that HCECs treatment with pituitary adenylate cyclase–activating polypeptide (PACAP) following growth factors deprivation prevents their degeneration. However, the molecular mechanism mediating this effect has not been clarified, yet. Here, we have shown for the first time the expression of PACAP and its receptor (PAC1R) in human corneal endothelium and demonstrated that this peptide, selectively binding to PAC1R, induces epidermal growth factor receptor (EGFR) phosphorylation and the MAPK/ERK1/2 signaling pathway activation. In conclusion, our data have suggested that PACAP could represent an important trophic factor in maintaining human corneal endothelial integrity through EGFR transactivation. Therefore, PACAP, as well as epidermal growth factor and fibroblast growth factor, could co-operate to guarantee tissue physiological functioning by supporting corneal endothelial barrier integrity.     

Effect of Intraocular Forward Scattering and Corneal Higher-Order Aberrations on Visual Acuity after Descemet’s Stripping Automated Endothelial Keratoplasty

Purpose

To assess the relationship of intraocular forward scattering and corneal higher-order aberrations (HOAs) with best spectacle corrected visual acuity (BSCVA) after Descemet’s stripping automated endothelial keratoplasty (DSAEK), and to compare these parameters between DSAEK and non-Descemet’s stripping automated endothelial keratoplasty (n-DSAEK) groups.

Methods

This retrospective study enrolled thirty eyes of 30 consecutive patients who underwent standard DSAEK, and who underwent successful phacoemulsification with intraocular lens implantation before DSAEK. The mean age at the time of surgery was 71.7 ± 10.4 years. We quantitatively evaluated the objective scattering index (OSI) using the double-pass instrument (OQAS II, Visiometrics) and corneal HOAs using Hartmann-Shack aberrometry (KR-9000PW, Topcon) 3 months postoperatively.

Results

The mean OSI, corneal HOAs, and logMAR BSCVA 3 months after DSAEK were 7.91 ± 3.58, 0.43 ± 0.27 μm, and 0.32 ± 0.25, respectively. We found a significant correlation between the OSI and logMAR BSCVA (Spearman correlation coefficient r=0.714, p<0.001), but no significant association between corneal HOAs and logMAR BSCVA 3 months postoperatively (r=0.209, p=0.267). We found no significant differences in any postoperative parameters between the DSAEK and n-DSAEK groups (p>0.05).

Conclusions

Our pilot study demonstrated that the postoperative corrected visual acuity was significantly correlated with intraocular forward scattering, but not with corneal HOAs in post-DSAEK eyes, suggesting that intraocular forward scattering plays a more essential role in postoperative visual performance than corneal aberrations after DSAEK. The detailed visual performance, such as HOAs and intraocular scattering, after n-DSAEK appears to be essentially equivalent to that after DSAEK.     

Polarization of human donor corneas

To investigate the de-orientation effect of DSAEK grafts by observing the cross patterns and polarization power of human donor corneas using a polarizing device (Lumaxis^®). Forty human donor corneas were placed in small petri-plates with epithelial side facing up. Polarizing power (arbitrary unit) and crosses were monitored and recorded by the software. The tissue was marked at ‘Superior’ position to ensure that the base and the polarizer are in alignment with each other after the cut. The anterior lamellar cut was performed using microkeratome. The lenticule was placed back in the same position as marked to mimic the alignment. The tissue was further rotated by 45° ensuring that the base of the cornea and the polarizer were in alignment. The polarization power and ‘crosses’ were identified at each step. The average of forty corneas from pre-cut to post-45° angular change showed statistically significant difference (p < 0.05) in terms of polarizing power. The cross-shaped pattern deformed and lost the sharpness towards 45° angle. However, multiple variances in terms of ‘cross-patterns’ were observed throughout the study. Lumaxis^® was able to determine the worst quality tissue in terms of polarization (no black zone and crosses). Despite the quality of cross pattern which can be used as an additional objective parameter to evaluate the optical properties of the corneal tissue, this preliminary study needs to be further justified in terms of clinical relevance whether polarization changes with oriented or de-oriented grafts have any effects and consequences on the visual acuity.     

Different characteristics of endothelial cells from central and peripheral human cornea in primary culture and after subculture

Summary Several methods for isolation and cultivation of human corneal endothelial cells have been described during the last few decades. In contrast to the situation in vivo, the cultured cells show mitogenic activity but often lose their typical morphological appearance. In this paper, we describe a technique to isolate and cultivate morphologically unchanged endothelium from the human cornea. This method revealed different characteristics of endothelial cells according to their position within the human cornea. Endothelial cells isolated from the central part have a morphology similar to that of cells in vivo (i.e., they are densely packed and show no mitogenic activity). In contrast, endothelial cells derived from the peripheral part of the cornea are characterized by mitogenic activity but their cell-to-cell attachment seems to be less tight than in vivo. The significance of these two different endothelial cell types for wound healing in the human cornea is discussed.     

Age‐related dystrophic changes in corneal endothelium from DNA repair–deficient mice

The corneal endothelium (CE) is a single layer of cells lining the posterior face of the cornea providing metabolic functions essential for maintenance of corneal transparency. Adult CE cells lack regenerative potential, and the number of CE cells decreases throughout life. To determine whether endogenous DNA damage contributes to the age‐related spontaneous loss of CE, we characterized CE in Ercc1^?/Δ mice, which have impaired capacity to repair DNA damage and age prematurely. Eyes from 4.5‐ to 6‐month‐old Ercc1^?/Δ mice, age‐matched wild‐type (WT) littermates, and old WT mice (24‐ to 34‐month‐old) were compared by spectral domain optical coherence tomography and corneal confocal microscopy. Histopathological changes in CE were further identified in paraffin tissue sections, whole‐mount immunostaining, and scanning electron and transmission electron microscopy. The CE of old WT mice displayed polymorphism and polymegathism, polyploidy, decreased cell density, increased cell size, increases in Descemet's thickness, and the presence of posterior projections originating from the CE toward the anterior chamber, similar to changes documented for aging human corneas. Similar changes were observed in young adult Ercc1^?/Δ mice CE, demonstrating spontaneous premature aging of the CE of these DNA repair–deficient mice. CD45⁺ immune cells were associated with the posterior surface of CE from Ercc1^?/Δ mice and the tissue expressed increased IL‐1α, Cxcl2, and TNFα, pro‐inflammatory proteins associated with senescence‐associated secretory phenotype. These data provide strong experimental evidence that DNA damage can promote aging of the CE and that Ercc1^?/Δ mice offer a rapid and accurate model to study CE pathogenesis and therapy.     

Rupture of Descemet's membrane secondary to presumed forceps trauma

Ruptures in Descemet's membrane can occur secondary to forceps injury at birth, prolonged labor, blunt trauma, keratoconus, and congenital glaucoma as well as other less frequent causes. The rupture initially causes acute comeal hydrops which resolves within weeks to leave permanent linear thickening of Descemet's membrane. It is important to differentiate Descemet's rupture from other entities like syphilitic interstitial keratitis, posterior polymorphous dystrophy and congenital hereditary endothelial dystrophy which may have a similar clinical presentation. Although treatment is rarely required, proper diagnosis is important to prevent unnecessary referrals and provide appropriate counseling. This case report examines a 66-year-old female with Descemet's rupture secondary to presumed forceps trauma at birth. Discussion of clinical presentation, differential diagnosis and clinical management are presented.     

Preparation of pre-cut corneas from fresh donated whole globes for Descemet’s stripping automated keratoplasty: 3-year results at the Central Eye Bank of Iran

To describe the technique and the results of the preparation of pre-cut corneas for Descemet’s stripping automated endothelial keratoplasty (DSAEK) during a 3-year period at the Central Eye Bank of Iran (CEBI). The method of preparation of pre-cut corneas from donated whole globes at the CEBI is described and the frequency and percentage of pre-cut corneas prepared for DSAEK, between April 2009 and March 2012, are specified. Moreover, post-operative reports are reviewed for any complaints about using pre-cut tissues for DSAEK. Out of the 1,518 donated whole globes appropriate for DSAEK, 1,478 (97.4 %) pre-cut corneas were successfully prepared. The method of preparation failed in 40 (2.6 %) cases. Based on the eye bank post-operative reports, thickness of pre-cut tissues for DSAEK was deemed unacceptable in only 6 (0.4 %) cases prior to surgery; five of these were too thick and one was too thin. Preparation of pre-cut corneas, for DSAEK from donated whole globes, in the CEBI is a safe and easy method, with very good preservation of endothelial cells after the preparation of the pre-cut corneas and reduced risks from corneal manipulation.     

Origin of Endothelium in Human Renal Allografts

Sex chromatin counts performed on the endothelial cells of 40 human kidneys transplanted to recipients of the opposite sex showed that the donor endothelium had persisted except in three poorly functioning and severely damaged grafts. In these a high proportion of the endothelial cells in peritubular capillaries and veins were derived from the host. Endothelial repopulation of organ allografts probably occurs only after severe tissue injury, and it cannot explain the phenomenon of graft adaptation. Repopulated endothelium may be derived from circulating cells.     

Clinical cryobiology of tissues: preservation of corneas

It is well recognized that the clarity of the cornea is a function of its hydration, and that this hydration is controlled by a "pump-and-leak" mechanism operating across the posterior monolayer of cells called the endothelium. A breakdown of the endothelium through disease or injury causes a marked increase in corneal thickness as the stroma imbibes fluid from the aqueous humor in the anterior chamber of the eye. This thickened, edematous condition of the stroma results in a cloudy cornea with an associated marked decrease in visual acuity. Treatment for this condition is usually by full-thickness corneal transplantation (penetrating keratoplasty), the success of which is dependent upon the donor cornea having an intact and healthy endothelium. It is essential, therefore, that any method of corneal storage for penetrating keratoplasty should protect and preserve the endothelium in a viable state. Current clinical practice relies upon short-term methods of preservation by two principal methods. Moist Chamber Storage is the time-honored corneal preservation method; it consists of keeping enucleated eyes at 0-4 degrees C in a sealed jar containing a pad of cotton gauze soaked in saline to provide a humid environment. The time limit placed upon this method of storage is 24-48 hr after which the viability of the endothelium deteriorates rapidly. Storage in M-K (McCarey-Kaufman) Medium involves excision of the corneoscleral segment from the donor eye and immersing it, endothelial side uppermost, in a medium consisting of tissue culture medium, 5% Dextran 40, and antibiotics. Laboratory and clinical studies indicate that storage in M-K medium at 4 degrees C preserves human endothelial cells for up to 4 days when the eye has been removed from the cadaver in less than 10 hr postmortem. Long-term preservation of corneas by freezing has long been a major goal in eye banking because indefinite storage by cryopreservation offers significant advantages for the quality and the quantity of material for use in keratoplasty, as well as for its distribution. However, procedures that have been developed for the cryopreservation of corneas have not been widely used, and a number of studies have shown that these procedures are inadequate for maintaining the integrity of the corneal endothelium. Not surprisingly, clinicians are now reluctant to accept corneas that have been frozen by these methods, though the clinical need is now greater than ever.(ABSTRACT TRUNCATED AT 400 WORDS)     

A dataset of human cornea proteins identified by Peptide mass fingerprinting and tandem mass spectrometry

Diseases of the cornea are extremely common and cause severe visual impairment worldwide. To explore the basic molecular mechanisms involved in corneal health and disease, the present study characterizes the proteome of the normal human cornea. All proteins were extracted from the central 7-mm region of 12 normal human donor corneas containing all layers: epithelium, Bowman's layer, stroma, Descemet's membrane, and endothelium. Proteins were fractionated and identified using two different procedures: (i) two-dimensional gel electrophoresis and protein identification by MALDI-MS and (ii) strong cation exchange or one-dimensional SDS gel electrophoresis followed by LC-MS/MS. All together, 141 distinct proteins were identified of which 99 had not previously been identified in any mammalian corneas by direct protein identification methods. The characterized proteins are involved in many processes including antiangiogenesis, antimicrobial defense, protection from and transport of heme and iron, tissue protection against UV radiation and oxidative stress, cell metabolism, and maintenance of intracellular and extracellular structures and stability. This proteome study of the healthy human cornea provides a basis for further analysis of corneal diseases and the design of bioengineered corneas.     

Suture Pull-Through Insertion Techniques for Descemet Stripping Automated Endothelial Keratoplasty in Chinese Phakic Eyes: Outcomes and Complications

Purpose

To investigate the outcomes and complications of suture pull-through insertion techniques for Descemet stripping automated endothelial keratoplasty (DSAEK) in Chinese phakic eyes.

Patients and Methods

Retrospective case series. Included in the study were all Chinese patients with phakic eyes who underwent DSAEK at Peking University Third Hospital from August 2008 to August 2011. All ocular diseases of the patients were recorded. Distance visual acuity (DVA), near visual acuity (NVA), intraocular pressure (IOP), anterior chamber depth (ACD), central corneal thickness (CCT), and corneal endothelial cell density (ECD) were compared prior to and 12 months after DSAEK. The DSAEK success rate, endothelial cell loss (ECL), complications, and prognosis were analyzed. All patients had at least 12 months of follow up.

Results

Twenty-one eyes of 16 patients were included (11 males and 5 females). Ages ranged from 2 to 47 years with an average age of 29.8 years. The average follow up was 15.4 months (ranging from 12 to 36 months). Diagnoses included 7 eyes (4 patients) with corneal endothelial dystrophy and 14 eyes (12 patients) with bullous keratopathy. Presurgical DVA and NVA (LogMAR) were 1.7±0.7 and 1.2±0.4; postsurgical DVA and NVA were 0.8±0.6 and 0.7±0.5; Z = −3.517, −2.764; P<0.001 and P = 0.006 respectively. Presurgical IOP was 15.8±3.7 mm Hg; postsurgical IOP was 15.2±2.6 mm Hg; Z = −0.505, P = 0.614. Presurgical ACD was 3.00±0.74 mm; postsurgical ACD was 2.72±0.59 mm; Z = −0.524, P = 0.600. Donor ECD was 2992±163 cells/mm², ECD was 1836±412 cells/mm² with a 12-month postsurgical ECL of 39%. Success rate was 86%. Surgery complications included pupillary block-induced hypertension in 5 eyes (24%), graft detachment in 3 eyes (14%), and graft dislocation in 1 eye (5%).

Conclusions

DSAEK on Chinese phakic eyes can significantly improve DVA and NVA by preserving the patient’s own crystalline lens. DSAEK is an optional surgery for patients who need to preserve accommodative function. More attention should be given to postsurgical pupillary block-induced hypertension.     

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g. endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca²⁺ signaling, which leads to production of diffusible autacoids (e.g. nitric oxide and arachidonic acid metabolites)^1-3 and hyperpolarization^4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca²⁺ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions^6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.     

内皮祖细胞在炎症损伤修复中的作用和机制

内皮祖细胞（endothelial progenitor cells,EPCs）是出生后,可以在机体内分化为成熟内皮细胞的一种前体细胞,主要来源于骨髓。多种伴有血管内皮细胞损伤的疾病都可引起外周血EPCs数量变化。有研究显示EPCs参与炎性损伤修复,并且外周血EPCs数量与血管内皮损伤程度和疾病预后存在一定的相关关系。EPCs。通过动员、迁移、归巢和分化等步骤修复内皮。炎症反应中受损组织释放的基质细胞衍生因子、血管内皮生长因子可与EPCs相应的受体结合,通过内皮型一氧化氮合酶、基质金属蛋白酶9等途径调节内皮修复过程,这是EPCs分化为内皮细胞过程的主要调控机制。此外,EPCs还可通过旁分泌机制促进相邻的内皮细胞增殖分化。目前,EPCs在炎症领域仅用于内皮炎性损伤和疾病预后评估,但是EPCs在心血管疾病和组织工程领域应用研究的成功,为EPCs在炎症反应的诊断和治疗提供了新的思路。